A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase
A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase
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DOI：
10.1111/j.1462-2920.2005.00854.x
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发表时间：
2005-12-01
影响因子：
5.1
通讯作者：
Johnston, AWB
中科院分区：
文献类型：
--
作者：
Wexler, M;Bond, PL;Johnston, AWB
利用从污水处理厂厌氧消化器宏基因组中获得的DNA，我们构建了一个克隆于广泛宿主cosmid pLAFR3的基因文库。其中一种微生物使豆科根瘤菌能够以乙醇为唯一的碳和能量来源生长，这是由于一种名为adhE（Meta）的基因的存在。AdhE（Meta）蛋白与乙酰丁酸梭菌（Clostridium acetobutylicum）的AdhE醇脱氢酶最相似，它在两步还原过程中催化乙醇和丁醇的形成。然而，克隆的adhE（Meta）并没有赋予大肠杆菌或铜绿假单胞菌利用乙醇的能力，尽管它在这两种宿主中都有转录。此外，对大肠杆菌和豆科致病菌的无细胞提取物进行体外丁醇脱氢酶和乙醇脱氢酶活性测定。与已被充分研究的C. acetobutylicum和E. coli的AdhE蛋白相反，AdhE所指定的酶（Meta）不会被氧灭活，它使酒精能够被分解代谢。然而，克隆的adhE Meta确实赋予了大肠杆菌一种表型。大肠杆菌的AdhE（-）突变体不能发酵葡萄糖，在发酵条件下，引入AdhE Meta可以恢复这些突变体的生长。这些观察结果表明，使用广泛的宿主载体可以提高筛选具有新功能基因的宏基因组文库的效率。
Using DNA obtained from the metagenome of an anaerobic digestor in a waste water treatment plant, we constructed a gene library cloned in the wide host-range cosmid pLAFR3. One cosmid enabled Rhizobium leguminosarum to grow on ethanol as sole carbon and energy source, this being due to the presence of a gene, termed adhE(Meta). The AdhE(Meta) protein most closely resembles the AdhE alcohol dehydrogenase of Clostridium acetobutylicum, where it catalyses the formation of ethanol and butanol in a two-step reductive process. However, cloned adhE(Meta) did not confer ethanol utilization ability to Escherichia coli or to Pseudomonas aeruginosa, even though it was transcribed in both these hosts. Further, cell-free extracts of E. coli and R. leguminosarum containing cloned adhE Meta had butanol and ethanol dehydrogenase activities when assayed in vitro. In contrast to the well-studied AdhE proteins of C. acetobutylicum and E. coli, the enzyme specified by adhE(Meta) is not inactivated by oxygen and it enables alcohol to be catabolized. Cloned adhE Meta did, however, confer one phenotype to E. coli. AdhE(-) mutants of E. coli fail to ferment glucose and introduction of adhE Meta restored the growth of such mutants when grown under fermentative conditions. These observations show that the use of wide host-range vectors enhances the efficacy with which metagenomic libraries can be screened for genes that confer novel functions.