(Re)design of the choroplast genome - towards a synthetic organelle

叶绿体基因组的（重新）设计 - 走向合成细胞器

• 批准号: BB/R01860X/1
• 负责人: Alison Smith
• 金额: $ 51.28万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR01860X%2F1
• 关键词: Re; design; choroplast; genome; towards

Plants and algal cells contain a compartment (or organelle) not found in animal cells - the chloroplast. This is the site of photosynthesis and other important biosynthetic processes, and it contains its own genetic system that is a legacy of the chloroplast's evolution from a free-living photosynthetic bacterium. Over evolutionary time, the circular genome of the chloroplast (the 'plastome') has been massively reduced in size, with the loss of most of its genes. What remains is a tiny genome that contains only a hundred-or-so genes. About half of these encode components of the photosynthetic apparatus, whilst the remainder are genes for housekeeping functions such as gene expression. The plastome therefore represents a naturally reduced genome that could be readily re-designed using synthetic biology approaches to gain insights into minimal requirements for an entire genome. At the same time this would optimize the plastome as a platform (a chassis) for future engineering efforts, such as production of high value products in the chloroplast or re-engineering the photosynthesis process. Genetic engineering of the plastome is well-established for several plant species, and for the single-celled green alga Chlamydomonas reinhardtii, which has served for many years as a model system for studying chloroplast biology. C. reinhardtii is particularly suited for a plastome redesign project as, unlike plant cells, it contains just a single chloroplast and can dispense completely with photosynthesis when grown on acetate as a source of carbon. Furthermore, the generation of chloroplast-engineered strains takes weeks rather than months. Recently, we have developed new tools for engineering the C. reinhardtii plastome and will apply these to address the following questions: i) by systematically deleting all regions of the plastome known to contain photosynthetic genes and other dispensable DNA, can we define the minimal size and gene content for the plastome? ii) Can we re-introduce the genes for a particular photosynthetic complex as a single refactored gene cluster, thereby allowing a modular 'plug-and-play' approach to studying how gene changes influence photosynthetic performance. iii) Can large gene clusters be engineered into the plastome to allow the reprogramming of the chloroplast as a site for synthetic of high-value products such as vitamin B12? iv) can we design and build an entirely synthetic minimal plastome and introduce this into the chloroplast, replacing the native plastome and thereby 'rebooting' the DNA software of the organelle? v) can we integrate into our plastome technology the capacity to tune up or down the expression of target genes using different combinations of chemicals in the growth medium, allowing us to control gene clusters or test pairs of gene variants in the same chloroplast by switching expression from one to the other?The project will provide essential basic understanding of the challenges of synthetic reprogramming of organelle genomes in plants and animal cells, and serve as a platform for future "designer organelle" studies.

植物和藻类细胞含有动物细胞中没有的区室（或细胞器）——叶绿体。这是光合作用和其他重要生物合成过程的场所，它包含自己的遗传系统，是叶绿体从自由生活的光合细菌进化而来的遗产。随着进化时间的推移，叶绿体的环状基因组（“质体”）的大小已大幅减小，大部分基因都丢失了。剩下的只是一个微小的基因组，仅包含一百个左右的基因。其中大约一半编码光合作用机构的组成部分，而其余的则是基因表达等管家功能的基因。因此，质体代表了一个自然减少的基因组，可以使用合成生物学方法轻松地重新设计，以深入了解整个基因组的最低要求。同时，这将优化质体作为未来工程工作的平台（底盘），例如在叶绿体中生产高价值产品或重新设计光合作用过程。质体基因工程在多种植物物种和单细胞绿藻莱茵衣藻中已经成熟，多年来它一直作为研究叶绿体生物学的模型系统。莱茵衣藻特别适合质体重新设计项目，因为与植物细胞不同，它仅含有单个叶绿体，并且在以醋酸盐作为碳源生长时可以完全省去光合作用。此外，叶绿体工程菌株的产生需要数周而不是数月的时间。最近，我们开发了用于工程莱茵衣藻质体的新工具，并将应用这些工具来解决以下问题：i）通过系统地删除已知含有光合基因和其他可有可无的 DNA 的质体的所有区域，我们能否定义最小尺寸以及质体的基因含量？ ii）我们能否将特定光合复合体的基因作为单个重构基因簇重新引入，从而允许采用模块化“即插即用”方法来研究基因变化如何影响光合性能。 iii) 是否可以将大型基因簇工程化到质体中，从而将叶绿体重新编程为合成高价值产品（如维生素 B12）的位点？ iv) 我们能否设计和构建一个完全合成的最小质体并将其引入叶绿体中，取代天然质体，从而“重新启动”细胞器的 DNA 软件？ v) 我们能否将在生长培养基中使用不同化学物质组合来调节目标基因表达的能力整合到我们的质体技术中，从而使我们能够通过切换表达来控制基因簇或测试同一叶绿体中的基因变体对从一种到另一种？该项目将为植物和动物细胞中细胞器基因组的合成重编程的挑战提供必要的基本了解，并作为未来“设计细胞器”研究的平台。

项目成果

Exploring the impact of terminators on transgene expression in Chlamydomonas reinhardtii with a synthetic biology approach

用合成生物学方法探索终止子对莱茵衣藻转基因表达的影响

• DOI: http://dx.10.1101/2021.08.04.455025
• 发表时间: 2021
• 作者: Geisler K

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism.

藻类叶绿体作为合成生物学设计的试验台，旨在从根本上重新布线植物代谢。

• DOI: http://dx.10.17863/cam.77032
• 发表时间: 2021
• 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

Exploring the Impact of Terminators on Transgene Expression in Chlamydomonas reinhardtii with a Synthetic Biology Approach.

用合成生物学方法探索终止子对莱茵衣藻转基因表达的影响。

• DOI: http://dx.10.3390/life11090964
• 发表时间: 2021
• 期刊: Life (Basel, Switzerland)
• 作者: Geisler K

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.84537
• 发表时间: 2022
• 作者: Jackson H