Kim Nam Hee, Park Ji-Hye, Chung Eunsook, So Hyun-Ah, Lee Myung Hwan, Kim Jin-Cheol, Hwang Eul Chul, Lee Seon-Woo
Department of Applied Bioscience, Dong-A University, Busan 49315, Republic of Korea.
Department of Agricultural Chemistry, Chonnam National University, Gwangju 61186, Republic of Korea.
J Microbiol Biotechnol. 2016 Feb;26(2):248-54. doi: 10.4014/jmb.1507.07029.
A soil metagenome contains the genomes of all microbes included in a soil sample, including those that cannot be cultured. In this study, soil metagenome libraries were searched for microbial genes exhibiting lipolytic activity and those involved in potential lipid metabolism that could yield valuable products in microorganisms. One of the subclones derived from the original fosmid clone, pELP120, was selected for further analysis. A subclone spanning a 3.3 kb DNA fragment was found to encode for lipase/esterase and contained an additional partial open reading frame encoding a wax ester synthase (WES) motif. Consequently, both pELP120 and the full length of the gene potentially encoding WES were sequenced. To determine if the wes gene encoded a functioning WES protein that produced wax esters, gas chromatography-mass spectroscopy was conducted using ethyl acetate extract from an Escherichia coli strain that expressed the wes gene and was grown with hexadecanol. The ethyl acetate extract from this E. coli strain did indeed produce wax ester compounds of various carbon-chain lengths. DNA sequence analysis of the full-length gene revealed that the gene cluster may be derived from a member of Proteobacteria, whereas the clone does not contain any clear phylogenetic markers. These results suggest that the wes gene discovered in this study encodes a functional protein in E. coli and produces wax esters through a heterologous expression system.
土壤宏基因组包含土壤样本中所有微生物的基因组，包括那些无法培养的微生物。在本研究中，对土壤宏基因组文库进行搜索，以寻找具有脂解活性的微生物基因以及那些参与潜在脂质代谢且能在微生物中产生有价值产物的基因。从原始fosmid克隆pELP120衍生的一个亚克隆被选用于进一步分析。发现一个跨度为3.3 kb DNA片段的亚克隆编码脂肪酶/酯酶，并且包含一个额外的部分开放阅读框，编码蜡酯合酶（WES）基序。因此，对pELP120和潜在编码WES的基因全长进行了测序。为了确定wes基因是否编码产生蜡酯的功能性WES蛋白，使用来自表达wes基因并与十六醇一起培养的大肠杆菌菌株的乙酸乙酯提取物进行了气相色谱 - 质谱分析。该大肠杆菌菌株的乙酸乙酯提取物确实产生了各种碳链长度的蜡酯化合物。全长基因的DNA序列分析表明，该基因簇可能源自变形菌门的一个成员，而该克隆不包含任何明确的系统发育标记。这些结果表明，本研究中发现的wes基因在大肠杆菌中编码一种功能性蛋白，并通过异源表达系统产生蜡酯。
