CAREER: Dissecting a Metabolically Versatile Non-Model Bacterium's Lignin-Derived Compound Catabolism

职业：剖析代谢多功能的非模型细菌的木质素衍生的复合分解代谢

• 批准号: 1943310
• 负责人: Rajib Saha
• 金额: $ 74.79万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1943310&HistoricalAwards=false
• 关键词: CAREER; Dissecting; Metabolically; Versatile; Non

This project investigates the potential causal factors behind a microbe’s ability to adapt to a wide range of conditions (e.g., presence/absence of oxygen) and carbon sources (e.g., compounds derived from lignin). If an enzyme involved in breaking down a carbon source is efficient but expensive to synthesize, in terms of energy, the reaction step it catalyzes may be rate limiting. This rate limiting step may dictate if or to what extent the microbe is able to live on the specific carbon source or whether it needs to have a secondary carbon source to survive. The project reveals the specific metabolic pathways (i.e., genes and enzymes) involved in breaking down a specific carbon source in the presence or absence of oxygen. A computer model is generated to predict the rate limiting step(s) in this process. These predictions are experimentally investigated to probe the role of specific genes under optimal growth conditions. The results of this project will enable biotechnologists to predictably and efficiently convert one of the most abundant waste products (i.e., lignin) to useful bioproducts (e.g., a biodegradable polymer). In addition, a major goal of this project is to improve the public perception of science literacy through a well-developed education platform targeting all audiences (from preschoolers to retired adults). The education activities include designing a series of biology books as well as interactive activities for preschoolers and mentoring middle/high school and undergraduate students to advance STEM education. Graduate students are trained broadly in the integrated nature of computation-driven experimentation to address relevant biological questions. In order to improve support and awareness for science, public lectures and interactive activities/demonstrations are also organized. This project addresses how a metabolically versatile purple non-sulfur bacterium, Rhodopseudomonas palustris, controls the catabolic pathways of lignin-derived compounds and connects these pathways with other biological processes. Although existing literature shows it has five annotated pathways (comprising of enzymes of broad substrate specificities) for catabolizing these compounds, how or if the bacterium is able to catabolize some of the major lignin-derived compounds or even polymeric lignin is unknown. There is also a lack of understanding of how sensitive the enzymes are in the presence/absence of oxygen and if there is a need for complementation by a secondary carbon source. The goal of this project is to integrate computational modeling with experimental approaches to address these critical gaps, and consequently unlocking the fundamental rules that allow the bacterium to adapt to a wide range of conditions and substrates. Relative contributions of catabolic reactions and associated genes/proteins are determined through a metabolism and expression (ME) model. This model is developed from available genome annotations and information derived from transcriptomics and quantitative proteomics data that will be obtained in this project. A synthetic biology toolbox, including inducible promoters, will be used to probe the role of key/limiting genes in pathways of interest. Findings from this project contribute to iterative ME model improvement through design-build-test-refine cycles for a more quantitative and mechanistic understanding of lignin-derived compound catabolism in microbes. In addition the project will be the vehicle for improving the public perception of science literacy, and developing education platform targeting to a wide range of audiences, from preschoolers to retired adults.This project is jointly funded by the Systems and Synthetic Biology cluster in the Division of Molecular and Cellular Biosciences and the Established Program to Stimulate Competitive Research (EPSCoR).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.

该项目调查了微生物适应各种条件（例如，氧的存在/不存在）和碳源（例如，衍生自木质素的化合物）。如果参与分解碳源的酶是有效的，但合成成本高，就能量而言，它催化的反应步骤可能是限速的。该速率限制步骤可以决定微生物是否或在多大程度上能够以特定碳源生存，或者它是否需要具有第二碳源才能生存。该项目揭示了特定的代谢途径（即，基因和酶）参与在有氧或无氧的情况下分解特定碳源。生成计算机模型以预测该过程中的速率限制步骤。这些预测进行实验研究，以探测特定基因在最佳生长条件下的作用。该项目的结果将使生物技术专家能够可预测地和有效地转化最丰富的废物之一（即，木质素）转化为有用的生物产品（例如，可生物降解的聚合物）。此外，该项目的一个主要目标是通过一个针对所有受众（从学龄前儿童到退休成年人）的完善的教育平台，提高公众对科学素养的认识。教育活动包括为学龄前儿童设计一系列生物学书籍以及互动活动，并指导初中/高中和本科生推进STEM教育。研究生在计算驱动的实验的综合性质，以解决相关的生物学问题进行了广泛的培训。 为了提高对科学的支持和认识，还组织了公开讲座和互动活动/示范。该项目解决了代谢多功能的紫色非硫细菌，沼泽红球藻，如何控制木质素衍生化合物的分解代谢途径，并将这些途径与其他生物过程连接起来。尽管现有文献显示其具有用于分解代谢这些化合物的五种注释途径（包括具有广泛底物特异性的酶），但是细菌如何或是否能够分解代谢一些主要的木质素衍生化合物或甚至聚合木质素是未知的。也缺乏对酶在存在/不存在氧的情况下的敏感性以及是否需要通过次级碳源进行补充的理解。该项目的目标是将计算建模与实验方法相结合，以解决这些关键差距，从而解锁允许细菌适应各种条件和底物的基本规则。通过代谢和表达（ME）模型确定分解代谢反应和相关基因/蛋白质的相对贡献。这个模型是从现有的基因组注释和信息来自转录组学和定量蛋白质组学数据，将在这个项目中获得。一个合成生物学工具箱，包括诱导型启动子，将用于探测关键/限制基因在感兴趣的途径中的作用。该项目的研究结果有助于通过设计-构建-测试-精炼循环迭代ME模型改进，以更定量和更机械地了解微生物中木质素衍生的化合物催化剂。此外，该项目还将成为提高公众对科学素养的认识和开发面向广泛受众的教育平台的工具，从学龄前儿童到退休成年人。该项目由分子和细胞生物科学部的系统和合成生物学集群以及刺激竞争研究的既定计划（EPSCoR）共同资助。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。

项目成果

Characterizing the interplay of rubisco 1 and nitrogenase enzymes in anaerobic-photoheterotrophically grown Rhodopseudomonas palustris CGA009 through a genome-scale metabolic and expression model

通过基因组规模代谢和表达模型表征厌氧光异养生长的沼泽红假单胞菌 CGA009 中 rubisco 1 和固氮酶的相互作用

• DOI: 10.1101/2022.03.03.482919
• 发表时间: 2022
• 期刊: bioRxiv
• 作者: Chowdhury, Niaz B.;Alsiyabi, A.;Saha, R.
• 通讯作者: Saha, R.

Biotechnical applications of phasins: Small proteins with large potential

• DOI: 10.1016/j.rser.2022.112129
• 发表时间: 2022-01-15
• 期刊: RENEWABLE & SUSTAINABLE ENERGY REVIEWS
• 影响因子: 15.9
• 作者: Brown，Brandi;Immethun，Cheryl;Saha，Rajib
• 通讯作者: Saha，Rajib

Protocol to develop a synthetic biology toolkit for the non-model bacterium R. palustris.

• DOI: 10.1016/j.xpro.2023.102158
• 发表时间: 2023-04-26
• 期刊: STAR PROTOCOLS
• 作者: Kathol, Mark;Immethun, Cheryl;Saha, Rajib
• 通讯作者: Saha, Rajib

Dissecting the metabolic reprogramming of maize root under nitrogen-deficient stress conditions

• DOI: 10.1093/jxb/erab435
• 发表时间: 2021-09-23
• 期刊: JOURNAL OF EXPERIMENTAL BOTANY
• 影响因子: 6.9
• 作者: Chowdhury, Niaz Bahar;Schroeder, Wheaton L.;Saha, Rajib
• 通讯作者: Saha, Rajib

Synergistic experimental and computational approach identifies novel strategies for polyhydroxybutyrate overproduction

• DOI: 10.1016/j.ymben.2021.08.008
• 发表时间: 2021-09-03
• 期刊: METABOLIC ENGINEERING
• 影响因子: 8.4
• 作者: Alsiyabi, Adil;Brown, Brandi;Saha, Rajib
• 通讯作者: Saha, Rajib