肠道微生物宏基因组文库构建及酯解酶基因筛选

利用宏基因组学方法,以人肠道微生物样品为原材料,构建了1个约30 000个克隆的fosmid文库.以三丁酸甘油酯为底物,通过功能筛选,获得1个酯解酶阳性克隆.对该阳性克隆构建亚克隆,挑选具有酯解酶活性的阳性亚克隆进行测序分析,最终获得1个肠道微生物来源的酯解酶基因(GenBank登录号:JQ972699).结果表明,文库克隆的平均插入片段约为40 kb,没有重复插入片段克隆.获得的酯解酶基因推演蛋白与Pyramidobacter piscolens W5455的patatin样磷脂酶同源性最高,氨基酸一致性为95％.生物信息学分析结果表明该基因可能通过Vd型分泌方式进行分泌并发挥功能.本研究是通过构建人肠道微生物宏基因组大片段文库并结合重组子功能筛选获得酯解酶的首次报道,可为食品工业提供新的酯解酶来源和筛选方法.

Screening of lipolytic genes from the metagenomic library of human intestine microbes

Metagenomic DNA was extracted from a human fecal sample and DNA fragments around 40 kb was isolated and purified.A fosmid library containing approximately 30 000 clones was created.Restriction analysis by EcoR I revealed a high diversity of the cloned DNA fragments and the estimated average insert size of the library was 40 kb.Tributyrin was used as substrate for activity-based screening of the library and one lipolytic enzyme positive fosmid clone was obtained.The lipolytic gene sequence was obtained after subcloning and sequencing,which showed high homology to the patatin-like phospholipase from Pyramidobacter piscolens W5455 with an amino acid identity of 95%.This is the first report of function-based metagenomics on screening of lipolytic genes from human gut microbiota.It also provides food industry with a new source of lipolytic genes and screening method.