PDF(Pubmed)
Abstract：
Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis, the microbial metabolism responsible for nearly all biological methane emissions to the atmosphere. Decades of biochemical and structural research studies have generated detailed insights into MCR function in vitro, yet very little is known about the interplay between MCR and methanogen physiology. For instance, while it is routinely stated that MCR catalyzes the rate-limiting step of methanogenesis, this has not been categorically tested. In this study, to gain a more direct understanding of MCR\'s control on the growth of Methanosarcina acetivorans, we generate a strain with an inducible mcr operon on the chromosome, allowing for careful control of MCR expression. We show that MCR is not growth rate-limiting in substrate-replete batch cultures. However, through careful titration of MCR expression, growth-limiting state(s) can be obtained. Transcriptomic analysis of M. acetivorans experiencing MCR limitation reveals a global response with hundreds of differentially expressed genes across diverse functional categories. Notably, MCR limitation leads to strong induction of methylsulfide methyltransferases, likely due to insufficient recycling of metabolic intermediates. In addition, the mcr operon is not transcriptionally regulated, i.e., it is constitutively expressed, suggesting that the overabundance of MCR might be beneficial when cells experience nutrient limitation or stressful conditions. Altogether, we show that there is a wide range of cellular MCR concentrations that can sustain optimal growth, suggesting that other factors such as anabolic reactions might be rate-limiting for methanogenic growth.
OBJECTIVE: Methane is a potent greenhouse gas that has contributed to ca. 25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. Methyl-coenzyme M reductase (MCR) catalyzes methane formatio in methanogens. Even though MCR comprises ca. 10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. In this study, we show that Methanosarcina acetivorans cells grown in substrate-replicate batch cultures produce more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher-throughput manner than isolation and biochemical characterization of pure protein.
OBJECTIVE: Methane is a potent greenhouse gas that has contributed to ca. 25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. Methyl-coenzyme M reductase (MCR) catalyzes methane formatio in methanogens. Even though MCR comprises ca. 10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. In this study, we show that Methanosarcina acetivorans cells grown in substrate-replicate batch cultures produce more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher-throughput manner than isolation and biochemical characterization of pure protein.
摘要：
甲基辅酶M还原酶（MCR）催化产甲烷的最后一步，微生物代谢负责几乎所有生物甲烷排放到大气中。数十年的生化和结构研究已经产生了详细的见解MCR功能在体外，然而，对MCR和产甲烷菌生理学之间的相互作用知之甚少。例如,虽然通常认为MCR催化产甲烷的限速步骤，这没有经过明确的测试。在这项研究中,为了更直接地了解MCR对甲烷藻生长的控制，我们产生了一个在染色体上具有可诱导的mcr操纵子的菌株，允许仔细控制MCR表达。我们表明，MCR在底物充足的分批培养中不限制生长速率。然而,通过仔细滴定MCR表达，可以获得生长限制状态。经历MCR限制的M.activorans的转录组学分析揭示了具有跨不同功能类别的数百个差异表达基因的全球反应。值得注意的是,MCR限制导致甲硫醚甲基转移酶的强诱导，可能是由于代谢中间体回收不足。此外,mcr操纵子不受转录调节，即，它是组成型表达的，这表明，当细胞经历营养限制或应激条件时，MCR的过量可能是有益的。总之,我们表明，有一个广泛的细胞MCR浓度，可以维持最佳的生长，这表明其他因素，如合成代谢反应，可能是产甲烷生长的限速因素。
目的：甲烷是一种强效的温室气体，25％的全球变暖在后工业时代。大气中的甲烷主要来源于生物，主要由称为产甲烷菌的微生物产生。甲基辅酶M还原酶（MCR）催化产甲烷菌中的甲烷形成。即使MCR包含ca.10%的细胞蛋白质组，据推测，在产甲烷过程中，它是生长受限的。在这项研究中,我们表明，在底物重复分批培养中生长的甲烷气细胞产生的MCR比其细胞对最佳生长的需求更多。本研究中概述的工具可用于以比纯蛋白质的分离和生化表征更高通量的方式完善MCR中甲烷生成和测定病变的代谢模型。
目的：甲烷是一种强效的温室气体，25％的全球变暖在后工业时代。大气中的甲烷主要来源于生物，主要由称为产甲烷菌的微生物产生。甲基辅酶M还原酶（MCR）催化产甲烷菌中的甲烷形成。即使MCR包含ca.10%的细胞蛋白质组，据推测，在产甲烷过程中，它是生长受限的。在这项研究中,我们表明，在底物重复分批培养中生长的甲烷气细胞产生的MCR比其细胞对最佳生长的需求更多。本研究中概述的工具可用于以比纯蛋白质的分离和生化表征更高通量的方式完善MCR中甲烷生成和测定病变的代谢模型。
