(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一个在动物细胞中没有的隔室（或细胞器）-叶绿体。这是光合作用和其他重要的生物合成过程的场所，它包含自己的遗传系统，这是叶绿体从自由生活的光合细菌进化而来的遗产。在进化过程中，叶绿体的环状基因组（“质体”）的大小已经大大减少，大部分基因都丢失了。剩下的是一个很小的基因组，只包含100个左右的基因。这些基因中大约有一半编码光合作用器的组成部分，而其余的是管家功能的基因，如基因表达。因此，质体组代表了一个自然减少的基因组，可以很容易地使用合成生物学方法重新设计，以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。质体的基因工程在几种植物物种中已经得到了很好的应用，单细胞的绿色衣藻莱茵衣藻（Chlamyeliumreinhardtii）多年来一直是研究叶绿体生物学的模型系统。C.莱茵衣藻特别适合于质体组重新设计项目，因为与植物细胞不同，它仅包含单个叶绿体，并且当在乙酸盐作为碳源上生长时可以完全免除光合作用。此外，叶绿体工程菌株的产生需要数周而不是数月。最近，我们已经开发了新的工具，工程的C。reinhardtii质体，并将应用这些来解决以下问题：i）通过系统地删除所有已知含有光合基因和其他叶绿体DNA的质体区域，我们可以定义质体的最小尺寸和基因含量吗？ii）我们能否重新引入特定光合复合物的基因作为单个重构基因簇，从而允许模块化的“即插即用”方法来研究基因变化如何影响光合性能。iii）大的基因簇能否被工程化到质体中，以允许叶绿体的重编程作为合成高价值产品（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，取代天然质体，从而“重启”细胞器的DNA软件？v）我们是否可以将利用培养基中不同化学物质组合来调节靶基因表达的能力整合到我们的质体组技术中，从而使我们能够通过将表达从一个切换到另一个来控制基因簇或测试同一叶绿体中的基因变体对？该项目将提供对植物和动物细胞中细胞器基因组合成重编程挑战的基本了解，并作为未来“设计器”研究的平台。

项目成果

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

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

• DOI: 10.3389/fpls.2021.708370
• 发表时间: 2021
• 期刊: Frontiers in plant science
• 影响因子: 5.6
• 作者: Jackson HO;Taunt HN;Mordaka PM;Smith AG;Purton S
• 通讯作者: Purton S

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

• DOI: 10.3390/life11090964
• 发表时间: 2021-09-14
• 期刊: Life (Basel, Switzerland)
• 作者: Geisler K;Scaife MA;Mordaka PM;Holzer A;Tomsett EV;Mehrshahi P;Mendoza Ochoa GI;Smith AG
• 通讯作者: Smith AG

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: 10.1101/2021.08.04.455025
• 发表时间: 2021
• 作者: Geisler K

Exploring the Impact of Terminators on Transgene Expression in

探索终止子对转基因表达的影响

• DOI: 10.17863/cam.77525
• 发表时间: 2021
• 作者: Geisler K