2021-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/706000目前有許多文獻證明氧化三甲胺 (Trimethylamine N-oxide, TMAO)和冠狀動脈疾病的發生有密切關係，而氧化三甲胺是由腸道產生的三甲胺 (trimethylamine, TMA)在肝臟代謝所產生。我們的合作團隊利用口服肉鹼耐量試驗 (oral carnitine challenge test, OCCT)發現112個受試者糞便檢體中，約有一半為高氧化三甲胺產生者 (high-TMAO producer)，而另一半則不會 (low-TMAO producer)，分析其腸道菌叢，在高氧化三甲胺產生者有 25% 腸道菌有 Emergencia timonensis，有 24% 腸道菌有Ihubacter massiliensis，但尚未針對low-TMAO producer相關菌種進行深入分析。因此本計畫擬分析這112個受試者的腸道菌叢，找出在low-TMAO producer中存在率最高的幾種菌株。另外也會分析腸道菌有 E. timonensis或I. massiliensis的受試者中low-TMAO producer的腸道菌叢，找出可能抑制三甲胺形成的菌種。分離出的菌種可和E. timonensis或I. massiliensis共同培養，並以高效液相層析儀(High performance liquid chromatography, HPLC)分析γ-丁基甜菜鹼(γ-butyrobetaine, γBB) 合成三甲胺的路徑是否受抑制。計畫預計完成: (1) 將以全基因體序列分析抑制三甲胺形成的菌種，選殖並剔除可能抑制三甲胺的基因； (2) 以跳躍子 (Transposon) 建構突變基因庫 (mutant library) 找出失去抑制三甲胺能力的突變株。本實驗室已成功建構一株厭氧菌Odoribacter splanchnicus的突變基因庫。另外，初步發現有一株菌種約可抑制E. timonensis超過40％三甲胺合成。High-TMAO producer相關菌種的發現有助於機制的了解，而low-TMAO producer相關菌種的發現則有應用的潛力。 There are several reports indicate a high association between trimethylamine N-oxide (TMAO) and development of coronary artery disease (CAD). TMAO is mainly produced from trimethylamine (TMA) by gut bacteria and formed TMAO in the liver. Our collaborators used the oral carnitine challenge test (OCCT) and found half of the 112 human fecal samples before and after carnitine supplementation were grouped as high-TMAO producer, whereas the others were grouped as low-TMAO producer. Emergencia timonensis accounted for 25% of gut microbiota from the high-TMAO producer and Ihubacter massiliensis accounted for 24% of gut microbiota from the high-TMAO producer. However, low TMAO producers-associated microbes were not analyzed. Thus, we aim to analyze the microbiome of these 112 human fecal samples and identify low TMAO producers-associated microbes with high abundance. In addition, we further to analyze the microbiome of low TMAO producers with E. timonensis or I. massiliensis in their gut microbiota and isolate TMA-inhibiting microbes. Low TMAO producers-associated microbes or TMA-inhibiting microbes will be co-cultured with E. timonensis or I. massiliensis to determine whether these microbes could inhibit γ-butyrobetaine convert to TMA, which was detected by high performance liquid chromatography. We plan: (1) to analyze the whole genome sequences of TMA-inhibiting microbes and then to delete their potential TMA-inhibiting genes, (2) to generate transposon mutant library and identify mutants with loss of TMA-inhibiting ability. We have already generated a mutant library in an anaerobe Odoribacter splanchnicus. Our preliminary data found one anaerobe could inhibit over 40% of TMA formation from E. timonensis. High TMAO producer-associated microbes are useful for understanding of TMA metabolism mechanism; low TMAO producer-associated microbes are potential for clinical application.冠狀動脈疾病γ-丁基甜菜鹼三甲胺腸道菌叢coronary artery disease (CAD)γ-butyrobetaine (γBB)trimethylamine (TMA)gut microbiota