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

重写遗传密码：藻类质体作为基础和应用研究的试验台

• 批准号: BB/W003538/1
• 负责人: Saul Purton
• 金额: $ 400.91万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW003538%2F1
• 关键词: Rewriting; Genetic; Code; Algal; Plastome

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.

所有细胞生命都使用通用遗传密码，提供基因与其蛋白质输出之间的信息联系。该密码有 64 个密码子，可将 20 个氨基酸组装成蛋白质，每个氨基酸有 1 到 6 个密码子。但尽管经过三十亿年的进化，密码子的分配基本上保持不变。这就提出了几个基本问​​题：i）遗传密码是早期优化的产物，还是与密码子表的其他排列同样可行的“冻结事故”？理论分析表明代码经过优化以最大限度地减少错误，但替代安排尚未经过实验测试。 ii) 是否可以通过重新分配一个或多个密码子来扩展密码以包括目前蛋白质中未发现的其他氨基酸（所谓的 ncAA）？迄今为止，此类工程仅在有限程度上实现。 iii) 工业生物技术能否利用具有重新排列和扩展代码的细胞来制造具有新功能的含 ncAA 的蛋白质（例如具有更长半衰期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担忧，因为它们将使用任何其他生物体都无法读取的代码？“基因组重新编码生物体”的创建代表着一项极具挑战性的努力，即使考虑到“简单”细胞，例如大肠杆菌。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，但它们的质体拥有独立的遗传系统，其基因组很小，只有一百个左右的基因，称为质体。使用单细胞藻类衣藻的合成生物学和基因工程的最新进展现在为使用该模型系统进行基因组重新编码、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领域的领先专家组成的联盟、基因组重新编码和 ncAA、植物合成生物学以及藻类基因工程和基因编辑。我们将开展一项雄心勃勃的工作计划，有五个主要目标： 1. 我们将生成一个合成质体组 (SynPlast1.0)，其中所有非必需基因均已被去除，其余基因使用最少的 51 个密码子集。这将演示密码子压缩的原理，并作为系统重新设计的基础。 2. 建立了质体设计和递送的管道后，我们将重新分配不同氨基酸的密码子以制作SynPlast2.0。然后，我们将根据细胞及其质体的生物学特性来评估包含这种新遗传密码的细胞的“适应性”。3。我们将利用步骤 1 中释放的备用密码子来扩展遗传密码以包含 ncAA。我们将证明可以将多个 ncAA 添加到代码中，并且可以合成具有新特性的蛋白质。4。为了将工程质体开发为亚细胞工厂，我们需要精确控制质体基因表达。我们将在细胞核中开发遗传开关，允许质体基因的可调表达。 5. 我们将利用藻类具有光合作用的事实来测试改善光合作用的不同重新设计策略。然后，我们将利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，其中结合了先前已被证明可赋予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、廉价且可持续地制造此类蛋白质。

项目成果

Over-expression of a cyanobacterial gene for 1-deoxy-d-xylulose-5-phosphate synthase in the chloroplast of Chlamydomonas reinhardtii perturbs chlorophyll: carotenoid ratios.

莱茵衣藻叶绿体中 1-脱氧-d-木酮糖-5-磷酸合酶蓝藻基因的过度表达会扰乱叶绿素与类胡萝卜素的比例。

• DOI: http://dx.10.1016/j.jksus.2022.102141
• 发表时间: 2022
• 期刊: Journal of King Saud University. Science
• 作者: Hoqani UA

CpPosNeg: A positive-negative selection strategy allowing multiple cycles of marker-free engineering of the Chlamydomonas plastome

CpPosNeg：一种正负选择策略，允许对衣藻质体进行多个循环的无标记工程

• DOI: http://dx.10.1002/biot.202200088
• 发表时间: 2022
• 期刊: Biotechnology Journal
• 影响因子: 4.7
• 作者: Jackson H

CpPosNeg: A positive-negative selection strategy allowing multiple cycles of marker-free engineering of the Chlamydomonas plastome.

CpPosNeg：一种正负选择策略，允许对衣藻质体进行多个循环的无标记工程。

• DOI: http://dx.10.17863/cam.84543
• 发表时间: 2022
• 作者: Jackson H

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts.

SAGA1 和 SAGA2 促进拟南芥叶绿体中原核蛋白周围淀粉的形成。

• DOI: http://dx.10.1073/pnas.2311013121
• 发表时间: 2024
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Atkinson N

Editorial: Structure and function of chloroplasts, Volume III.

社论：叶绿体的结构和功能，第三卷。

• DOI: http://dx.10.3389/fpls.2023.1145680
• 发表时间: 2023
• 期刊: Frontiers in plant science
• 作者: Gao H