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

人们对利用微藻作为工业生物技术的生产平台有相当大的兴趣，但这需要用于代谢工程的强大且可控的工具[2]。该项目的目的是利用内源性调控机制产生合成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，其中许多已被证明是细胞器转录物正常表达所需的RNA结合蛋白。在莱茵衣藻中，已知几种突变体在不同的TPR/PPR蛋白中具有缺陷，并且已经鉴定了它们的靶标，例如NAC 2（[1];参见轮换项目描述）。博士项目有三个目标：一。使用TPP-核糖开关调节的NAC 2基因来调节一个或多个引入到C.莱茵衣藻叶绿体基因组。一些化合物显示出细胞毒性，因此通过能够抑制基因直到细胞生长，这将避免问题。二.使用一种或多种其他TPR/PPR蛋白建立类似的调控回路，使用相同的核糖开关、对不同配体响应的修饰版本或诱导型启动子[3]。这两个电路的组合可以产生一个AND逻辑门（其中两个都必须起作用以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，诸如此类的合成生物学工具应该独立于环境进行操作，例如在不同的生物体中[2]。为了测试这一点，PPR/TPR蛋白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，以确定是否有任何共同点，或者它们是否都是物种特异性的。梭然后，将在一个有可能转化叶绿体基因组的物种中测试莱因哈德氏菌的遗传电路。目前，这对于其他藻类还没有很好地建立，但如果需要，可以在高等植物烟草（Nicotiana tabacum）（烟草）中进行。

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).