Multienzyme complexes of methanogenic archaea

产甲烷古菌（产甲烷菌）是“极端微生物”，在地球上生命极限的条件下茁壮成长的有机体。产甲烷菌是一种独特的生物体，通过产生甲烷气体来生长，甲烷气体可以用于电力，热能和运输燃料。该项目将研究一个大型多酶复合物的生物化学，类似于一个“氧化还原路由器”开关，能够根据细胞的能量状态将碳导向生物质或甲烷合成。据研究人员所知，这是在任何生物体中描述的第一个这样的生物路由器开关，它直接将能量守恒与生物质合成集成在一个酶复合物中。主要研究者将确定复合物中酶的比例，确定相互作用的位点，并确定复合物是否会因生长底物而改变组成。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。 研究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进行分享。这项研究有可能提高可再生能源的生物甲烷产量，减少环境中的甲烷产量，并通过合成生物学工程化产甲烷菌以生产有用的化学品。（CoM-S-S-CoB杂二硫还原酶，Hdr）和Wood-Ljungdahl CO2固定途径（一氧化碳脱氢酶Cdh，乙酰辅酶A脱羰酶/合酶ACDS的亚基;和亚甲基四氢甲烷蝶呤还原酶Mer）在产甲烷菌Methanosarcina acetivorans中形成多酶复合物。该项目的目标是确定产甲烷菌是否会调整Cdh/Hdr/Mer复合物的化学计量来响应生长基质的切换。假设M.乙酰化酶依赖于ACDS复合物的末端氧化还原酶Hdr与一氧化碳脱氢酶（Cdh）的形成和化学计量。分子，生物化学和生物物理技术将用于检测和表征在体外和体内形成的多酶复合物。将评估与生长底物相关的酶复合物组成和亚基交换动力学，并测量酶复合物组分的缺失和过表达对细胞生理学的影响。将采用体内和体外交联质谱法来绘制蛋白质相互作用界面，然后将其用于使用同源亚基的晶体结构来模拟路由器复合物。将操纵亚基化学计量，并确定其对细胞生长速率、产物产率和代谢效率的影响。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的培训。研究将通过出版物，演讲和推广活动，如流行的妇女在科学workshop.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.