2017年11月16日,David Liu(刘如谦)研究团队在Nature Chemical Biology 上发表了题为“Continuous directed evolution of aminoacyl-tRNA synthetases”的研究论文。该研究通过噬菌体辅助持续进化技术(PACE)对氨酰-tRNA合成酶(aminoacyl-tRNA synthetases,AARSs)进行持续定向进化,快速产生高活性和高选择性的正交AARSs.
酶分子的改造往往可以通过改变单个氨基酸残基来实现,然而,天然氨基酸的数量有限难以满足酶分子改造的需求。正交翻译系统(Orthogonal translation systems,OTSs)能够将非天然氨基酸(Noncanonical amino acids,ncAAs)特异性地整合到重组蛋白中,有超过200个非天然氨基酸已经通过OTSs被应用到蛋白质工程与修饰中。在向蛋白质引入ncAA的过程中,有四个组成部分至关重要,它们是:(1)ncAA本身;(2)编码ncAA的密码子(通常是TAG无意义密码子)被放置在编码序列中所需要的ncAA结合的位置;(3)一个不被宿主内源性AARSs识别的正交tRNA(o-tRNA),在翻译过程中解码无意义密码子;(4)将ncAA连接到tRNA上的AARS。在这四个组成部分中,开发出一个识别特定ncAA的正交AARS是最具挑战性的,利用传统定向进化方法改造获得新的AARS需要复杂的流程与筛选操作。
图1 正交翻译系统示意图
为了加速AARS的进化,刘如谦团队采用了噬菌体辅助连续进化(Phage-assisted continuous evolution,PACE)方法。PACE是由该团队开发出来的一种可以通过驾驭生物进化过程,产生具有指定功能的生物分子的技术。PACE选择系统使定向进化的速度比传统方法快100倍,实现了生物体“自发”的连续定向进化。
PACE系统是在连续流动的进化池中进行,研究人员将噬菌体侵染宿主菌所需的gIII基因切除,替换成待进化生物分子的基因。携带待进化野生型目的基因的噬菌体侵染宿主细胞时,将其遗传物质注入宿主菌,利用宿主菌内的复制系统进行遗传物质的复制。与此同时,在阿拉伯糖的诱导下,宿主细胞内诱变质粒上的基因表达,导致目的基因产生突变。若目的基因的突变可以启动gIII蛋白的表达,则可以产生具有侵染性的子代噬菌体。这些新产生的子代噬菌体突变株分泌到宿主菌外,感染新的宿主菌并进行下一轮复制增殖,提高自身数量,从而在进化池中存留下来。而无突变的噬菌体模块和不能启动gIII表达的突变噬菌体模块则不能分泌子代噬菌体进行增殖,其数量不会提高,最终从体系中被洗脱。
图2 PACE系统辅助的AARS进化示意图
研究人员将这种技术应用于吡咯赖氨酸氨酰-tRNA合成酶(PyIRS),进化出的PylRS变体具有提高了45倍的酶催化效率(kcat/KMtRNA)。
PACE系统不仅能进行正向筛选,还能进行反向或双向筛选。此外,PACE系统还可以被用来提高酶对底物的选择性,如PACE衍生的Methanocaldococcus jannaschii酪氨酰-tRNA合成酶(MjTyrRS)的嵌合突变体对底物p-碘-L-苯丙氨酸的特异性提高了23倍(注:图例中底物颜色标注反了)。
图3 PACE双向筛选提高AARS对底物的立体选择性
这项研究为提高AARS酶的催化效率和底物特异性提供了一个快速而有效的方法。通过ncAA扩展遗传密码,可以将一些对研究和治疗性产品开发有用的特征纳入蛋白质,加强对蛋白质结构与功能的理解,利用PACE方法改造AARS将发挥重要的作用。
论文链接:https://doi.org/10.1038/nchembio.2474
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