Weimar M R, Cheung J, Dey D, McSweeney C, Morrison M, Kobayashi Y, Whitman W B, Carbone V, Schofield L R, Ronimus R S, Cook G M
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
Rumen Microbiology, AgResearch Ltd., Palmerston North, New Zealand.
Appl Environ Microbiol. 2017 Jul 17;83(15). doi: 10.1128/AEM.00396-17. Print 2017 Aug 1.
Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H and CO that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen strain S2 and the rumen methanogen species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against and AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions. Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.
氢营养型产甲烷菌通常需要在玻璃管中进行严格厌氧培养，管内充有氢气和一氧化碳，这种培养条件既耗时又昂贵。为了提高筛选化合物文库的通量，开发了用于海洋（环境）产甲烷菌菌株S2和瘤胃产甲烷菌物种AbM4生长的96孔微量滴定板方法。对一些关键参数（接种量、培养基制备用还原剂、测定持续时间、抑制剂溶剂和培养体积）进行了优化，以实现高通量微量滴定板形式下稳健且可重复的生长。该方法使用已发表的产甲烷菌抑制剂进行了验证，并对其灵敏度和可重复性进行了统计学评估。作为实用性验证，针对包含1280种药理活性化合物的Sigma-Aldrich LOPAC文库和一个内部天然产物文库（120种化合物）进行了筛选。该筛选鉴定出了一些生物活性化合物，并对其中一些化合物针对菌株S2和AbM4的最低抑菌浓度（MIC）值进行了确认。所开发的方法显著提高了筛选化合物文库的通量，现在可用于筛选更大的化合物文库，以发现用于减少反刍动物甲烷排放的新型产甲烷菌特异性抑制剂。反刍动物的甲烷排放是全球温室气体排放的重要贡献者，农业技术（agritech）领域需要新技术来控制排放。利用针对化合物文库（合成和天然产物）的高通量表型（生长）筛选发现产甲烷菌的小分子抑制剂是一条有吸引力的途径。然而，目前表型抑制剂筛选受到我们无法以高通量形式培养产甲烷菌的阻碍。我们已经开发、优化并验证了一种用于培养环境和瘤胃产甲烷菌的高通量96孔微量滴定板测定法。利用这个平台，我们鉴定出了几种新的产甲烷菌生长抑制剂，证明了这种方法在快速推进用于控制反刍动物甲烷排放的产甲烷菌特异性抑制剂开发方面的实用性。
