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標題: 從土壤多源基因體中篩選出離胺酸消旋酵素並以離胺酸消旋酵素基因作為植物基因選殖的篩選標記
Isolation and Characterization of a Novel Lysine Racemase Gene from a Soil Metagenomic Library and Utilization lyr as a Selectable marker for plant transformation
作者: 陳怡潔
Chen, I-Chien
關鍵字: 離胺酸消旋酵素;lysine racemase;土壤多源體基因庫;篩選標記;metagenomic library;selection marker
出版社: 分子生物學研究所
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Racemase具有生物轉換特性,催化天然及非天然胺基酸之旋光性,轉換質子的移動,產生不同旋光性胺基酸產物。本研究利用土壤多源體基因庫,搭配高複製套數的載體以及lysine營養缺陷菌株 Escherichia coli BCRC 51734,藉由功能性互補的方式,搭配D-lysine為特定氮源,選殖出離胺酸消旋化酶 (lyr) 基因。lyr基因由393個胺基酸所組成,分子量為42.7 kDa。生物資訊分析顯示,Lyr蛋白C-端 (殘基 188-393)與E. coli K12 argC 基因N-acetyl-γ-glutamyl- phosphate reductase (NAGPR)蛋白的N-端 (殘基1-206)具有100 %的相似度。而Lyr蛋白的N-端 (殘基 1-187)與Deinococcus radiodurans ATCC 13939的N-acylamino acid racemase (NAAAR)蛋白的N-端 (殘基 1-187),則具有98.4 %的相似度。然而純化後的Lyr蛋白,卻不具有NAGPR以及NAAAR的酵素活性。分析Lyr酵素活性,其最適反應溫度及pH值分別為30oC與pH 8.0,若在反應中分別加入Co2+、Mg2+ 和Mn2+等二價金屬離子,則可提高酵素的反應活性。以ICP spectrometer分析Lyr蛋白,顯示每一莫耳的二具體蛋白分子中含有0.77莫耳的Zn2+金屬離子。此酵素具有很嚴格的基質專一性,只對lysine具有消旋化酶的催化活性,催化D型或L型lysine之消旋化作用,Lyr對於L型或D型lysine的specific activity分別為3.61 U/mg 及1.68 U/mg,kcat 值分別為0.085±0.003 以及0.036±0.002 min-1。本研究是首次從土壤多元基因體中分離選殖出lyr基因,並報導其基因序列。本研究中,利用lysine racemase (lyr)的生物轉換特性,以L-lysine作為篩選劑,作為植物基因轉形時之篩選標記,並成功應用於菸草及阿拉伯芥這兩種模式植物中。在植物對於胺基酸敏感性的研究中,發現植物對於L-lysine相當敏感,反之,對於D-lysine則無此現象。因此利用消旋化酶的生物轉換特性,將L-lysine轉換成D-lysine作為植物生長的氮源。發現,在Arabidopsis中利用lyr作為篩選標記,比傳統上使用kanamycin篩選的轉形效率高2倍,且轉形後的所得到的轉殖植物,其發芽速度、植物體質量、葉片數目及根的長度上,與野生型植物相比較並無顯著差異。另一方面,Lyr 蛋白在轉基因菸草植物中的表現活性為0.77 to 1.06 mU/mg protein,此外,比對轉殖菸草與野生型菸草的內生性胺基酸濃度,僅aspartic acid的濃度受到lyr表現的影響而改變,顯示,此基因對於內生性胺基酸的代謝之影響非常有限。值得注意的是,研究中發現植物的aspartate kinases可區別lysine的旋光性,僅受到L-lysine的回饋調控。本研究為首次報導利用lyr基因取代抗藥性基因,作為植物基因轉形時之篩選標記,使用於模式植物-Nicotiana benthamiana Domin. 及Arabidopsis,成功的獲得基因轉殖植物,此篩選標記未來將可廣泛被使用於D-lysine或L-lysine敏感的植物。

A lysine racemase (lyr) gene was isolated from a soil metagenomic library by functional complementation using Escherichia coli BCRC 51734, a lysine auxotrophic mutant, as host cell and D-lysine as selection agent. This lyr gene contained an open reading frame encoding a protein composed of 393 amino acids with a molecular mass of about 42.7 kDa. Bioinformatics analysis revealed that the C-terminal portion (residues 188-393) of lysine racemase (Lyr) showed 100% amino acid identity to the N-terminal portion (residues 1-206) of N-acetyl-γ-glutamyl- phosphate reductase (NAGPR) encoded by E. coli K12 argC gene and the N-terminal portion (residues 1-187) of Lyr displayed 98.4% identity to N-terminal portion (residues 1-187) of N-acylamino acid racemase (NAAAR) from Deinococcus radiodurans ATCC 13939. However, no NAGPR and NAAAR activities were found for purified Lyr. The temperature and pH optimal for the enzyme activity of the Lyr protein were determined as 30oC and pH 8.0, respectively. The enzyme activity was enhanced significantly by metal ions Co2+, Mg2+ and Mn2+. The dialyzed Lyr contained 0.77 mol of Zn2+ per mol of the dimeric enzyme as revealed by the ICP spectrometer assay. The enzyme exhibited higher specific activity toward lysine (3.61 U/mg protein versus 1.68 U/mg protein) as well as a higher kcat (0.085±0.003 versus 0.036±0.002 min-1) in the L-lysine to D-lysine direction as compared to the reversible reaction. This is the first report of cloning, purification and characterization of a novel lysine racemase gene from a soil metagenome.
In this study, a non-antibiotic based selection system using L-lysine as selective agent and the lysine racemase (lyr) as selectable marker gene to develop the transgenic plants in two model plant species viz., tobacco and Arabidopsis was established. In preliminary experiments showed that several plant species are sensitive to L-lysine, while D-lysine supports their growth. Therefore, we used Lyr to catalyze the racemization of L- to D-lysine which would be used by transgenic plants as nitrogen source. The efficiency of transformation was approximately two-fold higher than kanamycin selection in Arabidopsis. Transgenic tobacco and Arabidopsis plants selected on L-lysine exhibited normal growth characteristics as that of wild-type plants and grew vigorously upon transfer to soil. The specific activity of Lyr in transgenic tobacco plants selected on L-lysine ranged from 0.77 to 1.06 mU/mg protein, whereas no activity was virtually detectable in wild-type plants. In addition, the composition of the free amino acids, except aspartic acid, was not affected by the expression of lyr in the transgenic tobacco plants suggesting very limited interference with endogenous amino acid metabolism. Interestingly, our findings also indicated that the plant aspartate kinases may possess an ability to distinguish the enantiomers of lysine for feedback regulation. This is the first report of a novel selection marker for transformation in which lyr gene instead of drug-resistant gene is used as selectable marker in the genetic transformation of two model plant species viz., Nicotiana benthamiana Domin. and Arabidopsis using L-lysine as selection agent.
其他識別: U0005-0408200913004500
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