Rewriting The Genetic Code: The Algal Plastome As A Testbed For Basic And Applied Studies

所有的细胞生命都使用一种通用的遗传密码，该密码提供了基因及其蛋白质输出之间的信息联系。该密码有密码子，可以让20个氨基酸组装成蛋白质，每个氨基酸有1到6个密码子。但是，尽管经过了超过30亿年的进化，密码子的分配基本上保持不变。这引发了几个基本问题：i)遗传密码是早期优化的产物，还是与密码表的其他排列同样可行的“冻结意外”？理论分析表明，代码经过了优化，以最大限度地减少错误，但替代方案尚未进行实验测试。Ii)是否可以通过重新分配一个或多个密码子，将密码扩展到包括目前在蛋白质中未发现的其他氨基酸(所谓的NCAA)？到目前为止，这样的工程只在有限的程度上实现了。三)工业生物技术能否利用重新排列和扩展密码的细胞来制造具有新功能的含有NCAA的蛋白质(例如半衰期更长的激素药物或具有新催化功能的酶)？Iv)这种重新编码能否解决有关合成基因逃逸和水平转移的问题和公众关注的问题，因为它们将使用任何其他有机体都无法识别的密码？即使考虑到像大肠杆菌这样的“简单”细胞，创造“基因组重新编码的生物体”也是一项极具挑战性的工作。在这个项目中，我们将通过关注叶绿体(或‘叶绿体’)来降低这一挑战的复杂性。虽然藻类和植物细胞的大部分基因都在细胞核中，但它们的质体拥有一个自给自足的遗传系统，只有100个左右的基因组成的微小基因组被称为质体体。利用单细胞藻衣藻属的合成生物学和基因工程的最新进展为利用该模型系统进行基因组重组、扩展和开发提供了可能性。在这个项目中，我们汇集了叶绿体合成生物学、基因组重组和NCAA、植物合成生物学、藻类基因工程和基因编辑等领域的顶尖专家。我们将进行一项雄心勃勃的工作计划，主要有五个目标：1.我们将产生一个合成质体(SynPlast1.0)，其中所有非必需基因都已被移除，其余基因使用最少的51个密码子。这将证明密码子压缩的原理，并作为重新设计该系统的基础。2.在建立了质体组设计和交付的管道后，我们将把密码子重新分配到不同的氨基酸上，以制造SynPlast2.0。然后，我们将根据细胞及其胞质的生物学特性来评估含有这种新遗传密码的细胞的“适合性”。我们将利用第一步中释放的多余密码子将遗传密码扩展到包括NCAA。我们将演示可以在密码中添加多个NCAA，并可以合成具有新特性的蛋白质。为了将工程化质体体发展成为亚细胞工厂，我们需要精确地调控质体体基因的表达。我们将在细胞核中开发基因开关，使叶绿体基因的表达可调。5.我们将利用藻类具有光合作用的事实来测试不同的重组策略，以改善光合作用。然后，我们将使用该项目的集体知识来演示两种药物蛋白质的光驱动合成，结合以前已被证明具有有价值的新治疗特性的NCAA。这将使未来的技术能够利用二氧化碳和阳光简单、廉价和可持续地制造这种蛋白质。

All cellular life uses a universal genetic code that provides the informational link between genes and their protein outputs. The code has 64 codons to enable 20 amino acids to be assembled into proteins, with between 1 and 6 codons per amino acid. But the assignment of the codons has remained essentially unchanged despite over three billion years of evolution. This raises several fundamental questions: i) is the genetic code the product of early optimisation, or a 'frozen accident' with other arrangements of the codon table equally viable? Theoretical analyses suggest that the code is optimised to minimise errors, but alternative arrangements have not been tested experimentally. ii) Can the code be expanded to include other amino acids that are not currently found in proteins (so called ncAAs) through the reassignment of one or more codons? To-date, such engineering has been achieved only to a limited degree. iii) Could industrial biotechnology exploit cells with a rearranged and expanded code to make ncAA-containing proteins with novel features (e.g. hormone drugs with longer half-lives, or enzymes with new catalytic functions)? iv) Could such recoding address issues and public concerns around escape and horizontal transfer of synthetic genes given that they would use a code that is unreadable by any other organism?The creation of 'genome recoded organisms' represents an extremely challenging endeavour, even when considering a 'simple' cell such as E. coli. In this project we will reduce the complexity of this challenge by focussing on the chloroplast (or 'plastid'). Whilst algal and plant cells have most of their genes in the nucleus, their plastids possess a self-contained genetic system with a tiny genome of only a hundred-or-so genes known as the plastome. Recent advances in synthetic biology and genetic engineering using the single-cell alga Chlamydomonas now offer the potential for genome recoding, expansion, and exploitation using this model system.In this project we bring together a consortium of leading experts in the fields of chloroplast synthetic biology, genome recoding and ncAAs, plant synthetic biology, and algal genetic engineering and gene editing. We will undertake an ambitious programme of work with five main goals: 1. We will generate a synthetic plastome (SynPlast1.0) in which all non-essential genes have been removed and the remaining genes use a minimal set of 51 codons. This will demonstrate the principle of codon compression and also serve as the basis for redesign of the system. 2. Having establish the pipeline for plastome design and delivery, we will reassign codons to different amino acids to make SynPlast2.0. We will then assess the 'fitness' of cells containing this new genetic code in terms of the biology of the cell and its plastid.3. We will extend the genetic code to include ncAAs by making use of the spare codons released in step 1. We will demonstrate that multiple ncAAs can be added to the code, and that proteins with novel properties can be synthesised.4. In order to develop the engineered plastid as a sub-cellular factory, we need to precisely control plastome gene expression. We will develop genetic switches in the nucleus that allow the tuneable expression of plastid genes. 5. We will exploit the fact that the alga is photosynthetic to test different re-engineering strategies for improving photosynthesis. We will then use the collective knowledge from the project to demonstrate the light-driven synthesis of two pharmaceutical proteins, incorporating ncAAs that have previously been shown to confer valuable new therapeutic properties. This will allow future technology where such proteins are made simply, cheaply and sustainably using CO2 and sunlight.