Establishing the use of TPR/PPR proteins for synthetic biology of microalgae

开发微藻作为工业生物技术的生产平台有相当大的兴趣，但这需要强大和可控的代谢工程工具[2]。这个项目的目的是利用内源调节机制，产生合成的基因电路来控制转基因在藻类叶绿体中的表达。植物和藻类在细胞核中编码PPR(五肽重复)和TPR(四肽重复)蛋白的多个基因，其中许多被证明是细胞器转录本正常表达所需的RNA结合蛋白。在莱茵衣藻中，已知有几个突变体在不同的TPR/PPR蛋白中存在缺陷，并且已经确定了它们的靶标，如NAC2([1]；见旋转项目描述)。PHD项目有三个目标：1.使用TPP-核糖开关调控的NAC2基因来调节导入到莱茵哈迪叶绿体基因组中的类异戊二烯生物合成酶的一个或多个基因。一些化合物表现出细胞毒性，所以通过抑制基因直到细胞生长，就可以避免这个问题。二、使用一个或多个其他TPR/PPR蛋白，或者使用相同的核糖开关，或者使用对不同配体反应的修饰版本，或者使用可诱导的启动子，来建立类似的调节电路。这两个电路的组合可能导致AND逻辑门(两者都必须起作用才能获得叶绿体基因的表达)或OR门(其中只有一个必须起作用)。三、理想情况下，这样的合成生物学工具应该独立于环境而运行，例如在不同的生物体中[2]。为了验证这一点，将通过生物信息学和共表达分析在其他藻类基因组中鉴定PPR/TPR蛋白，以确定是否有任何共同之处，或者它们是否都是物种特有的。然后，将在一个有可能转化叶绿体基因组的物种中测试莱茵哈迪尔锥虫的遗传电路。目前，这还不适用于其他藻类，但如果有必要，可以在高等植物烟草(烟草)中实现这一点。

There is considerable interest in exploitation of microalgae as production platforms for industrial biotechnology, but this requires robust and controllable tools for metabolic engineering [2]. The aim of this project is to generate synthetic gene circuits to control expression of transgenes in the algal chloroplast, using endogenous regulatory mechanisms. Plants and algae encode multiple genes in the nucleus for PPR (pentatricopeptide repeat) and TPR (tetratricopeptide repeat) proteins, many of which have been shown to be RNA binding proteins required for normal expression of organelle transcripts. In Chlamydomonas reinhardtii, several mutants are known with defects in different TPR/PPR proteins, and their targets have been identified, such as NAC2 ([1]; see rotation project description). The PhD project has three objectives: i. Use the TPP-riboswitch-regulated NAC2 gene to regulate one or more genes for isoprenoid biosynthetic enzymes introduced into the C. reinhardtii chloroplast genome. Some of the compounds show cytotoxicity, so by being able to suppress the gene until the cells have grown this would avoid the problem. ii. Establish a similar regulatory circuit using one or more of the other TPR/PPR proteins, either using the same riboswitch, a modified version responsive to a different ligand, or an inducible promoter [3]. Combination of the two circuits could then result in an AND logic gate (where both must be functional to get expression of the chloroplast gene) or an OR gate (where only one must function). iii. Ideally, synthetic biology tools such as these should operate independently of context, such as in different organisms [2]. To test this, PPR/TPR proteins would be identified in other algal genomes by bioinformatics and co-expression analysis to establish if there are any in common, or whether they are all species-specific. The C. reinhardtii genetic circuits would then be tested in a species in which it is possible to transform the chloroplast genome. Currently this is not well established for other algae, but if necessary it could be done in the higher plant Nicotiana tabacum (tobacco).