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，在基因和它们的蛋白质输出之间提供信息联系。该密码有64个密码子，使20个氨基酸能够组装成蛋白质，每个氨基酸有1到6个密码子。但是密码子的分配尽管经过了30多亿年的进化，但基本上没有改变。这就提出了几个基本问题：1)遗传密码是早期优化的产物，还是密码子表的其他排列同样可行的“冻结事故”？理论分析表明，代码是优化的，以尽量减少错误，但其他安排还没有经过实验测试。ii)通过一个或多个密码子的重新分配，该密码子是否可以扩展到包括目前在蛋白质中未发现的其他氨基酸（所谓的ncAAs） ？到目前为止，这种工程只在有限的程度上得到了实现。iii)工业生物技术是否可以利用具有重排和扩展代码的细胞来制造具有新特征的含ncaa的蛋白质（例如半衰期较长的激素药物，或具有新催化功能的酶）？iv)考虑到合成基因会使用其他生物无法读取的代码，这种重新编码是否可以解决有关逃逸和水平转移的问题和公众担忧？即使考虑到像大肠杆菌这样的“简单”细胞，“基因组编码生物”的创造也是一项极具挑战性的努力。在这个项目中，我们将通过关注叶绿体（或“质体”）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独立的遗传系统，其基因组只有100个左右的基因，被称为质体。利用单细胞藻类衣藻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编码、扩展和开发提供了潜力。在这个项目中，我们汇集了叶绿体合成生物学、基因组重新编码和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。我们将开展一项雄心勃勃的工作方案，其中包括五个主要目标：我们将生成一个合成质体（SynPlast1.0），其中所有非必需基因已被删除，其余基因使用最小的51个密码子集。这将展示密码子压缩的原理，并作为重新设计系统的基础。2. 在建立了质体体设计和传递的管道后，我们将把密码子重新分配到不同的氨基酸上，制作SynPlast2.0。然后，我们将根据细胞及其质体的生物学特性来评估含有这种新遗传密码的细胞的“适应性”。我们将利用步骤1中释放的备用密码子扩展遗传密码以包含ncaa。我们将证明多个ncaa可以添加到代码中，并且可以合成具有新特性的蛋白质。为了使工程质体成为亚细胞工厂，我们需要精确控制质体基因的表达。我们将在细胞核中开发基因开关，使质体基因的可调表达成为可能。5. 我们将利用藻类进行光合作用的事实来测试不同的重新设计策略来改善光合作用。然后，我们将利用该项目的集体知识来演示两种药物蛋白的光驱动合成，其中包含之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简单、廉价和可持续地利用二氧化碳和阳光来制造这种蛋白质。

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.