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

植物和藻类细胞含有一种动物细胞所没有的细胞器——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，这是叶绿体从一个自由生活的光合细菌进化而来的遗产。在进化的过程中，叶绿体的环状基因组（“质体”）的大小已经大大缩小，失去了大部分基因。剩下的是一个很小的基因组，它只包含100个左右的基因。这些基因中大约有一半编码光合作用装置的成分，而其余的则是负责诸如基因表达等家务功能的基因。因此，质体体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，以获得对整个基因组的最低要求的见解。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。质体的基因工程在许多植物物种中已经建立，单细胞绿藻莱茵衣藻（Chlamydomonas reinhardtii）多年来一直是研究叶绿体生物学的模式系统。莱茵C. reinhardtii特别适合于质体重新设计项目，因为与植物细胞不同，它只包含一个叶绿体，当以醋酸盐作为碳源生长时，可以完全不进行光合作用。此外，叶绿体工程菌株的产生需要数周而不是数月。最近，我们开发了新的工具来设计C. reinhardtii质体，并将应用这些工具来解决以下问题：i)通过系统地删除所有已知的含有光合作用基因和其他必要DNA的质体区域，我们可以确定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体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作用性能？iii)能否将大的基因簇设计到质体中，以允许叶绿体重新编程，作为合成高价值产品（如维生素B12）的场所？iv)我们能否设计并构建一个完全合成的最小质体，并将其引入叶绿体，取代原生质体，从而“重启”细胞器的DNA软件？V)我们能否将质体技术整合到使用生长介质中不同化学物质组合来调节或降低目标基因表达的能力中，从而允许我们通过转换表达来控制基因簇或测试同一叶绿体中的基因变体对？该项目将为植物和动物细胞中细胞器基因组合成重编程的挑战提供必要的基本理解，并为未来的“设计细胞器”研究提供平台。

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.