Assessing secoisolariciresinol diglucoside metabolism in the rumen. Assessing secoisolariciresinol diglucoside metabolism in the rumen by means of a fosmid library

Secoisolariciresinol diglucoside, a lignan found in many plants, predominantly in flaxseed. The plant lignan can play active role enhancing antioxidant capacity, however, it need to be metabolized into enterolignans, process which the gut microbiota is responsible. The rumen is a known efficient environment for lignan metabolism, which comprises the breakdown of SDG into secoisolariciresinol, then to enterodiol and enterolactone. The product enterolactone increases concentration in the rumen as flax is added in the diet of ruminants and the compound can be transferred to milk, benefiting human health by the functional food intake, preventing many oxidative-related disorders such as cardiovascular diseases, atherosclerosis, Alzheimer’s disease, breast-cancer and others. Although the knowledge on lignans metabolism in the rumen, little information is available regarding the microorganisms linked to secoisolariciresinol diglucoside breakdown. The main bottleneck is that just few species of the rumen microbiome can be cultured in laboratory conditions, and thus, metagenomic approaches are needed to access the rumen ecology by-passing the in vitro culture. Therefore, a fosmid library was constructed using rumen microbes DNA inserts from the rumen of cows fed up to 15g/100g (dry matter basis) of flax meal, resulting on an 11,520 fosmid clones library, using Escherichia coli strain EPI300 as host bacteria. The library was screened for secoisolariciresinol diglucoside breakdown capacity using high-performance liquid chromatography and positive clones followed to plasmid DNA purification, deep sequencing and bioinformatic data analysis. Two out of 30 library plates showed effective secoisolariciresinol diglucoside breakdown, however, no metabolism products were identified and no putative gene linked to direct secoisolariciresinol diglucoside breakdown was identified from clones genome. To our knowledge this is the first fosmid library constructed to prospect rumen microorganisms with active role in plant lignans metabolism, thus, more studies regarding the subject must be carried out to broaden genes and enzymes involved in the process.

开环异落叶松脂素二葡萄糖苷（Secoisolariciresinol diglucoside，SDG）是一类广泛存在于多种植物中的木脂素，尤以亚麻籽中含量最为丰富。该植物木脂素可发挥增强抗氧化能力的活性作用，但其需经肠道菌群介导代谢为肠木脂素（enterolignans）后方可生效。瘤胃是已知的高效木脂素代谢环境，可将SDG依次降解为开环异落叶松脂素，随后转化为肠二醇（enterodiol）与肠内酯（enterolactone）。当日粮中添加亚麻籽时，反刍动物瘤胃内的肠内酯浓度会显著升高，且该物质可转移至牛乳中，通过功能性食品摄入为人类健康带来益处，有助于预防多种氧化相关疾病，如心血管疾病、动脉粥样硬化、阿尔茨海默病、乳腺癌等。尽管目前对瘤胃内木脂素的代谢已有一定认知，但与SDG降解相关的微生物信息却十分匮乏。主要瓶颈在于，仅有极少数瘤胃微生物组物种可在实验室条件下培养，因此需要借助宏基因组学方法绕过体外培养限制，以解析瘤胃微生态。本研究构建了宏基因组fosmid文库（fosmid library）：以饲喂干物质占比高达15g/100g亚麻籽粉的奶牛瘤胃微生物DNA为插入片段，以大肠杆菌EPI300（Escherichia coli strain EPI300）菌株作为宿主菌，最终获得包含11520个fosmid克隆的文库。通过高效液相色谱（high-performance liquid chromatography）筛选该文库中具有SDG降解能力的克隆，对阳性克隆进行质粒DNA纯化、深度测序及生物信息学数据分析。结果显示，30个文库平板中有2个平板呈现出有效的SDG降解活性，但未检测到代谢产物，也未从克隆基因组中鉴定出与SDG直接降解相关的推定基因。据我们所知，本研究是首个构建用于筛选具有植物木脂素代谢活性瘤胃微生物的fosmid文库，因此未来需开展更多相关研究，以拓展对该代谢过程所涉及基因与酶类的认知。