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，但这需要强大和可控的代谢工程工具。本项目的目的是利用内源性调控机制，生成合成基因回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（Chlamydomonas reinhardtii）中，已知几种突变体在不同的TPR/PPR蛋白中存在缺陷，并且已经确定了它们的靶点，例如NAC2（[1]；参见轮换项目描述）。博士项目有三个目标：1 .利用tpp -核糖体开关调控的NAC2基因调控一个或多个基因导入莱茵c.r ainhardtii叶绿体基因组的类异戊二烯生物合成酶。有些化合物显示出细胞毒性，所以通过抑制基因直到细胞生长，就可以避免这个问题。2。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，或诱导启动子[3]。这两个回路的结合可以产生一个与逻辑门（两者必须都有功能才能获得叶绿体基因的表达）或一个或门（只有一个必须起作用）。3。理想情况下，诸如此类的合成生物学工具应该独立于环境运行，例如在不同的生物体中运行。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，以确定它们是否有共同之处，或者它们是否都是物种特异性的。然后，莱茵梭菌的遗传回路将在一个有可能转化叶绿体基因组的物种中进行测试。目前还没有很好地建立其他藻类，但如果有必要，可以在高等植物烟草（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).