Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21525
標題: Utilization of lysien racemase gene as a selection marker for gene cloning
以離胺酸消旋酵素基因作為基因選殖的篩選標記
作者: 陳怡潔
Chen, Chien-I
關鍵字: 離胺酸消旋酵素;lysine racemase
出版社: 分子生物學研究所
摘要: 
Genetic engineering of microorganisms is highly dependent on the application of antibiotic resistance genes. Transfromants harboring antibiotic resistance genes may cause the risk of spread of antibiotic resistance traits to environmental microbes. In this study, we reported a novel selection marker for transformation in which lysine racemase (lyr)gene instead of drug-resistant gene is used as selectable marker and wild-type strains can be used as host cells for gene transformation.
Enzymatic assay of several racemases revealed that the enzymes lack lysine racemase activity. Two lysine racemase genes, lyr1 and lyr2, were obtained by error-prone PCR random mutagenesis using Amycolatopsis orientalis subsp. lurida N-acylamino acid racemase(aar)gene as template. ORFs of lyr1 and lyr2 genes with difference at residue 134 were 1,125 bp and coded a protein of 42 kDa. The segment of C-terminus(residues 175 to 375) showed 100 % identity to N-terminal segment of Escherichia coli argC gene(residues 1 to 201). N-terminal segment(residues 1 to 174)didn't show identify to any gene from organisms. Lyr can catalyze the racemization of L- and D-lysine. Plasmid harboring lyr gene was transformed into E. coli CCRC 30129, E. coli BL21(DE3), Aspergillus oryzae CCRC 30129, Bacillus subtilis, and Bacillus circulans and transformants could grow in the medium containing D-lysine as sole nitrogen source. Our data indicated that the lyr gene can be used as a selectable marker for the transformation of microorganisms.

現代生物科技的成功發展主要源於基因工程技術的進步,而基因工程技術的關鍵在於適當的篩選標記。因此,目前工業上大多利用抗藥性作為基因重組的非功能性互補篩選標記,但這些抗菌劑並非對每一種微生物皆有很好的抑制效果,有鑑於此非功能性互補的篩選基因有其迫切的需要性。本實驗的目的為利用lysine racemase的生物轉換特性,來轉換胺基酸的旋光性,作為宿主之氮源或必需營養成分,替代抗藥性基因,作為基因重組時之篩選標記。
分析數種racemases的活性,顯示這些racemases,不具有轉換lysine鏡像結構之能力。因此,以Amycolatopsis orientalis subsp. lurida N-acylamino acid racemase(aar)基因為模板,利用error-prone PCR進行隨機突變,以營養選擇性培養基進行篩選,總共得到2個突變菌株,分別命名為AarM1和AarM2,突變之racemase基因命名為lyr1和lyr2。lyr1和lyr2基因全長皆為1,125 bp,分子量為42 kDa,兩基因僅於第134胺基酸殘基有差異。由NCBI BLAST分析發現Lyr C-端(175到375殘基)與E. coli argC基因(1到201殘基)的相似度為100 ﹪,Lyr N-端(1到174殘基)無法比對出相似的微生物基因片段。Lyr具有轉換lysine鏡像結構之能力。
利用選殖在質體上的lyr1基因,搭配E. coli CCRC 30129、E. coli BL21(DE3)、Aspergillus oryzae CCRC 30129、Bacillus sibtilis 、及Bacillus circulans等不能利用D-lysine作為氮源的宿主細胞,以lyr1基因作為基因轉形的篩選標記,使轉形菌株能生長於以D-lysine作為氮源的培養基上。本研究結果顯示lyr1基因可作為微生物基因重組時之篩選標記。
URI: http://hdl.handle.net/11455/21525
Appears in Collections:分子生物學研究所

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