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.

所有的细胞生命都使用一个通用的遗传密码，它提供了基因及其蛋白质输出之间的信息联系。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，尽管经过了30亿年的进化，密码子的分配基本上没有变化。这提出了几个基本问题：i）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，代码进行了优化，以尽量减少错误，但替代安排尚未进行实验测试。ii）通过重新分配一个或多个密码子，可以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）吗？迄今为止，这种工程仅在有限程度上实现。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）这种重新编码是否可以解决围绕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都无法读取的代码？即使考虑像大肠杆菌这样的“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努力。杆菌在这个项目中，我们将通过关注叶绿体（或“质体”）来减少这一挑战的复杂性。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，只有一个只有100个左右基因的微小基因组，称为质体。利用单细胞衣原体进行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编码、扩增和开发提供了可能性。在本项目中，我们汇集了叶绿体合成生物学、基因组编码和ncAAs、植物合成生物学以及藻类基因工程和基因编辑领域的领先专家。我们将实施一项雄心勃勃的工作方案，其中有五个主要目标：1.我们将产生一个合成质体（SynPlast1.0），其中所有非必需基因已被删除，剩余的基因使用最少的51个密码子。这将证明密码子压缩的原理，并作为重新设计系统的基础。2.在建立了质体组设计和递送的管道之后，我们将重新分配密码子到不同的氨基酸以制造SynPlast2.0。然后，我们将根据细胞及其质体的生物学特性，评估含有这种新遗传密码的细胞的“适应性”。我们将通过利用第1步中释放的备用密码子来扩展遗传密码以包括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。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基因开关，允许质体基因的可调表达。5.我们将利用这一事实，即植物是光合作用，以测试不同的再造策略，以提高光合作用。然后，我们将利用该项目的集体知识来展示两种药物蛋白质的光驱动合成，并将先前已被证明具有有价值的新治疗特性的ncAA结合起来。这将使未来的技术能够使用二氧化碳和阳光简单，廉价和可持续地制造这些蛋白质。

项目成果

Spray Drying Is a Viable Technology for the Preservation of Recombinant Proteins in Microalgae.

• DOI: 10.3390/microorganisms11020512
• 发表时间: 2023-02-17
• 期刊: MICROORGANISMS
• 影响因子: 4.5
• 作者: Vilatte, Anaelle;Spencer-Milnes, Xenia;Jackson, Harry Oliver;Purton, Saul;Parker, Brenda
• 通讯作者: Parker, Brenda

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: 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.

• DOI: 10.1073/pnas.2311013121
• 发表时间: 2024-01-23
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Atkinson, Nicky;Stringer, Rhea;Mitchell, Stephen R.;Seung, David;McCormick, Alistair J.
• 通讯作者: McCormick, Alistair J.

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

• DOI: 10.1016/j.jksus.2022.102141
• 发表时间: 2022-08
• 期刊: JOURNAL OF KING SAUD UNIVERSITY SCIENCE
• 影响因子: 3.8
• 作者: Al Hoqani, Umaima;Leon, Rosa;Purton, Saul
• 通讯作者: Purton, Saul

ADA: an open-source software platform for plotting and analysis of data from laboratory photobioreactors

ADA：用于绘制和分析实验室光生物反应器数据的开源软件平台

• DOI: 10.1080/26388081.2021.2023632
• 发表时间: 2022
• 期刊: Applied Phycology
• 作者: Mapstone L