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Multienzyme complexes of methanogenic archaea

产甲烷古菌的多酶复合物

基本信息

批准号：
1938948
负责人：
Nicole Buan
金额：
$ 59.9万
依托单位：
University of Nebraska-Lincoln
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-15 至 2024-02-29
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1938948&HistoricalAwards=false
关键词：
Multienzyme complexes methanogenic archaea

项目摘要

Methane-producing archaea (methanogens) are “extremophiles”, organisms that thrive under conditions at the limit of life on Earth. Methanogens are unique organisms that grow by producing methane gas which can be harvested for electricity, heat, and transportation fuel. The project will study the biochemistry of a large multi-enzyme complex, akin to a “redox router” switch that has the ability to direct carbon towards either biomass or methane synthesis depending on the energy status of the cell. To the researchers' knowledge this is the first such biological router switch described in any organism that directly integrates energy conservation to biomass synthesis in one enzyme complex. The principal investigator will determine the ratio of enzymes in the complex, identify sites of interaction, and ascertain if the complex changes composition in response to growth substrate. A postdoctoral researcher and graduate students will be trained in the biochemical mechanisms of biological methane production. Research will be shared using hands-on educational modules through the Women in Science and other K-12 outreach activities. This research has the potential to enhance bio-methane production for renewable energy, mitigate methane production in the environment, and engineer methanogens to produce useful chemicals through synthetic biology.Previous work has shown that enzymes in the Wolfe Cycle (CoM-S-S-CoB heterodisulfide reductase, Hdr) and Wood-Ljungdahl CO2 fixation pathways (the carbon monoxide dehydrogenase Cdh, subunit of the acetyl-CoA decarbonylase/synthase, ACDS; and methylene tetrahydromethanopterin reductase, Mer) form a multienzyme complex in the methanogen Methanosarcina acetivorans. The goal of the project is to determine if methanogens adjust Cdh/Hdr/Mer complex stoichiometry in response to growth substrate switching. The hypothesis is that methanogenic growth kinetics of M. acetivorans is dependent on the formation and stoichiometry of the terminal oxidoreductase Hdr with the carbon monoxide dehydrogenase (Cdh) of the ACDS complex. Molecular, biochemical, and biophysical techniques will be used to detect and characterize multienzyme complexes that form in vitro and in vivo. Enzyme complex composition and subunit exchange kinetics will be assessed in relation to growth substrate, and the effect of deletion and overexpression of enzyme complex components on cell physiology will be measured. In vivo and in vitro crosslinking mass spectrometry will be employed to map protein interaction interfaces which will then be used to model the router complex using crystal structures of homologous subunits. Subunit stoichiometries will be manipulated and their effect on growth rate, product yield, and metabolic efficiency of cells will be determined. Graduate students and postdoctoral researchers from underrepresented groups will be trained in anaerobic microbiology, redox biochemistry, and synthetic biology techniques. Research will be disseminated through publications, presentations, and outreach activities such as the popular Women in Science workshop.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.

产生甲烷的古生菌(产甲烷菌)是“极端微生物”，它们在地球生命极限的条件下繁衍生息。产甲烷菌是一种独特的生物，它们通过产生甲烷气体来生长，甲烷气体可以被收集来发电、取暖和运输燃料。该项目将研究一种大型多酶复合体的生物化学，类似于“氧化还原路由器”开关，它能够根据细胞的能量状态将碳引导到生物质或甲烷合成中。据研究人员所知，这是在任何生物体中描述的第一个直接将能量守恒与生物质合成结合在一个酶复合体中的生物路由器开关。首席研究员将确定复合体中酶的比例，确定相互作用的位置，并确定复合体是否会随着生长底物的生长而改变组成。一名博士后研究员和研究生将接受生物甲烷生产的生化机制方面的培训。将通过妇女参与科学和其他K-12外联活动，使用实践教育模块分享研究成果。这项研究有可能提高可再生能源的生物甲烷产量，减少环境中甲烷的产生，并通过合成生物学改造产甲烷菌生产有用的化学物质。先前的工作表明，在产甲烷菌中，Wolfe循环中的酶(COM-S-S-cob异二硫化物还原酶)和Wood-Ljugdahl二氧化碳固定途径中的酶(乙酰辅酶A脱羧酶/合成酶的亚基一氧化碳脱氢酶CDH和亚甲基四氢甲烷五氢喋呤还原酶Mer)在产甲烷菌中形成多酶复合体。该项目的目标是确定产甲烷菌是否调整CDH/HDR/Mer复合体的化学计量比以响应生长底物的切换。假设醋酸菌的产甲烷生长动力学依赖于末端氧化还原酶HDR与ACDS复合体的一氧化碳脱氢酶(CDH)的形成和化学计量比。将使用分子、生化和生物物理技术来检测和表征在体外和体内形成的多酶复合体。将评估与生长底物有关的酶复合体组成和亚基交换动力学，并测量酶复合体组分的缺失和过表达对细胞生理的影响。体内和体外的交联质谱将被用来绘制蛋白质相互作用的界面，然后将被用来用同源亚基的晶体结构来模拟路由器复合体。亚基化学计量学将被操纵，它们对细胞生长速度、产物产量和代谢效率的影响将被确定。来自代表性不足群体的研究生和博士后研究人员将接受厌氧微生物学、氧化还原生物化学和合成生物学技术方面的培训。研究将通过出版物、演示文稿和推广活动传播，如受欢迎的女性参与科学工作坊。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。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Addressing the climate crisis through engineering biology

通过工程生物学应对气候危机

DOI：
10.1038/s44168-023-00089-8
发表时间：
2024
期刊：
npj Climate Action
影响因子：
0
作者：
Aurand, Emily R.;Moon, Tae Seok;Buan, Nicole R.;Solomon, Kevin V.;Köpke, Michael;EBRC Technical Roadmapping Working Group
通讯作者：
EBRC Technical Roadmapping Working Group

An Assessment of Short-Term Milestones in EBRC’s 2019 Roadmap, Engineering Biology

EBRC 2019 年路线图的短期里程碑评估，工程生物学

DOI：
发表时间：
2023
期刊：
https://roadmap.ebrc.org/2019-roadmap/an-assessment-of-engineering-biology-2023/.
影响因子：
0
作者：
Engineering Biology Research Consortium
通讯作者：
Engineering Biology Research Consortium

Metabolic Feedback Inhibition Influences Metabolite Secretion by the Human Gut Symbiont Bacteroides thetaiotaomicron