人肠道梭菌和甲烷马赛球菌协同代谢甜菜碱和胆碱产甲烷研究

Clostridium and Methanomassiliicoccus isolated from human intestine synergistically convert betaine and choline to methane

摘要:

【目的】根据人肠道富含胆碱和甜菜碱，同时肠道微生物组中具有裂解胆碱和还原甜菜碱产三甲胺的细菌，以及利用三甲胺产甲烷的古菌，本研究探讨肠道细菌与古菌协同代谢甜菜碱和胆碱产甲烷的可能性。【方法】调查不同年龄段人群粪便中的16S rRNA基因多样性，分析肠道中古菌的菌群组成；利用定量PCR (quantitative PCR, qPCR)定量甲烷马赛球菌(Methanomassiliicoccus)特异的甲醇甲基转移酶基因mtaB和甲烷八叠球菌(Methanosarcina)及细菌的16S rRNA基因拷贝数，分析肠道中甲基营养型产甲烷古菌及总细菌的含量；宏基因组组装基因组(metagenome-assembled genomes, MAGs)分析携带甜菜碱还原酶基因grdH和胆碱裂解酶基因cutC的细菌组成。从粪便中分离代谢甜菜碱及胆碱产生三甲胺的细菌，并与分离自人肠道的甲烷马赛球菌构建共培养物，测定其协同转化甜菜碱和胆碱产甲烷的能力。【结果】年轻人粪便中含有甲烷杆菌科(Methanobacteriaceae, 82.16%)的甲烷短杆菌属(Methanobrevibacter, 49.18%)和甲烷杆菌属(Methanobacterium, 33.34%)、甲基营养型的甲烷八叠球菌科(Methanosarcinaceae, 5.67%)的甲烷八叠球菌属(Methanosarcina, 5.70%)，以及甲烷马赛球菌科(Methanomassiliicoccaceae, 3.13%)的甲烷马赛球菌属(Methanomassiliicoccus, 3.14%)。而中老年人粪便中的甲烷古菌多样性较低，也未检测到甲烷马赛球菌。qPCR定量分析显示年轻人比中老年人肠道的总古菌含量高3.11倍，其中甲烷马赛球菌高6.53倍、甲烷八叠球菌高5.52倍，总细菌含量高2.90倍。宏基因组分析组装了229个细菌基因组，其中42个携带基因grdH和cutC，这些细菌属于毛螺菌科(Lachnospiraceae)、肠杆菌科(Enterobacteriaceae)和梭菌科(Clostridiaceae)等。从粪便中分离到恶名梭菌(Clostridium malenominatum) B8，菌株B8与卢米尼甲烷马赛球菌(Methanomassiliicoccus luminyensis) B10共培养物可降解47.03%的甜菜碱和25.83%胆碱，并产生甲烷，在培养液中检测到三甲胺先积累后被降解。【结论】人肠道细菌恶名梭菌B8和卢米尼甲烷马赛球菌B10可协同代谢甜菜碱和胆碱产甲烷，推测它们在人肠道中可将部分食物中的甜菜碱和胆碱代谢产生甲烷。

Abstract:

[Objective] To explore the feasibility of using Clostridium and Methanomassiliicoccus from human intestine to synergistically convert betaine and choline to methane. [Methods] Illumina sequencing of the 16S rRNA gene was performed to survey the diversity of archaea in the feces from healthy people of 20-40 years old and over 40 years old. The Methanomassiliicoccus-specific mtaB gene and Methanomassiliicoccus-specific 16S rRNA gene were quantitated by quantitative PCR (qPCR) to quantify the trimethylamine-utilizing methanogens in human intestine. Metagenome-assembled genomes (MAGs) were reconstructed from metagenome data for the identification of the intestinal bacteria carrying the betaine reductase gene grdH and choline trimethylamine-lyase gene cutC. The bacteria that reduced betaine and choline were isolated from feces and used to construct the coculture with Methanomassiliicoccus. The potential of the coculture for producing methane from betaine and choline was then determined. [Results] The main methanogenic archaea in the intestine of the 20-40 years old included Methanobrevibacter (49.18%) and Methanobacterium (33.34%) affiliating to Methanobacteriaceae (82.16%), Methanosarcina (5.70%) of Methanosarcinaceae (5.67%), and Methanomassiliicoccus (3.14%) of Methanosassilicoccaceae (3.13%). The methanogen diversity was lower in the feces from the people over 40 years old, from whom Methanosassilicoccaceae was not detected. Quantitative PCR determined that the total abundance of archaea and bacteria in the people of 20-40 years old was 3.11 and 2.90 folds, respectively, higher than in those over 40 years old. Specifically, the abundance of Methanomassiliicoccus and Methanosarcina was 6.53 and 5.52 folds higher, respectively. A total of 229 bacterial MAGs were obtained from the fecal specimens, in which 42 MAGs carried genes grdH and cutC and were affiliated to Lachnospiraceae, Enterobacteriaceae, and Clostridiaceae. Clostridium malenominatum B8 was isolated from the fecal specimens. The co-culture of this strain with Methanomassiliicoccus luminyensis B10 in the medium with 20 mmol/L betaine or choline degraded 47.03% betaine and 25.83% choline to produce methane, during which trimethylamine was detected as the intermediate. [Conclusion] The human intestinal Clostridium B8 and M. luminyensis B10 synergistically convert betaine and choline to methane. Therefore, we hypothesize that they play a role in reducing the trimethylamine in human intestine.