'21ENGBIO' Towards SYnthetic CHLOroPlastS (SYCHLOPS)

已经产生了具有完全或部分合成基因组的细菌和酵母，并且这些被证明是工程生物学的有用平台。例如，精心设计的合成基因组可以允许关键基因随意交换或重新排列。此外，同义密码子和UAG终止密码子已被重新分配，以允许扩展的遗传密码。一个很好的例子是E。大肠杆菌菌株Syn 61，其中所有丝氨酸UCG和UCA密码子被所有UAG终止密码子沿着替换。这一绝技允许这些密码子的重新分配，并将两个非规范氨基酸掺入由引入的转基因制成的蛋白质中。然而，酵母和细菌的基因组相对较大，创建完全或部分合成的版本具有挑战性，耗时且成本高昂。植物包含三个基因组，核，叶绿体（质体）和线粒体。核基因组是最大的，通常编码超过27，000个基因，使用130到几千兆碱基，具体取决于植物物种。相比之下，叶绿体基因组通常仅由15万个碱基对组成，其中120个基因紧密包装。然而，叶细胞可以含有超过100个叶绿体，每个叶绿体具有100个或更多个叶绿体基因组拷贝。因此，尽管叶绿体在细胞序列复杂性中所占的比例不到0.1%，但它可以贡献超过10%的DNA含量。部分原因在于，位于叶绿体基因组上的基因可以产生比位于核基因组上的等效单拷贝基因高得多的蛋白质水平（高达300倍）。此外，叶绿体被排除在花粉之外，并且叶绿体DNA仅遗传自授粉作物而不是授粉作物。这使得叶绿体作为“绿色工厂”非常有吸引力，用于生产新的高价值蛋白质、代谢物和生物聚合物，其中需要高水平的基因表达。此外，研究小组有兴趣通过操纵关键蛋白质来提高光合效率，例如重新设计CO2固定RuBisCO大亚基或用适应不同光环境的植物或藻类的大亚基取代它。然而，这样的实验目前超出了现有质体工程技术的能力。叶绿体编码核糖体和tRNA，这些是支持其基因组上编码序列的蛋白质合成所必需的（主要与管家功能和光合作用有关），但叶绿体中发现的大多数蛋白质（包括氨酰-tRNA合成酶和RNA pol亚基）在核基因组上编码并从细胞质输入。叶绿体基因组的直接转化在30年前首次实现，但由于难以引入大的DNA元件和确保转基因质体基因组完全取代未修饰的质体基因组的问题，因此在技术上仍然具有挑战性。如果试图用全合成序列取代基因组的大部分，也会存在类似的问题。我们最近使用双组分基因驱动系统和工程化的植物来解决其中的两个问题，这些植物含有单个巨大的叶绿体而不是>100个较小的叶绿体。利用这个系统，我们将构建和测试一个质体工程工具集，用于1）迭代引入大盒以引入复杂的生化途径，2）进行靶向基因组重排，最终允许质体基因组的大部分部分被定制的合成版本取代。

Bacteria and yeast with fully or partially synthetic genomes have been generated and these are proving to be useful platforms for engineering biology. For example, a well-designed synthetic genome can allow key genes to be swapped in and out or rearranged at will. In addition, synonymous codons and UAG stop codons have been reassigned to allow an expanded genetic code. A good illustration of this is the E. coli strain Syn61 in which all serine UCG and UCA codons were replaced along with all UAG stop codons. This tour de force allowed the re-assignment of these codons and the incorporation of two noncanonical amino acids into proteins made from introduced transgenes. However, yeast and bacterial genomes are relatively large and creating full or partially synthetic versions is challenging, time consuming and costly.Plants contain three genomes, the nuclear, the chloroplast (plastid) and the mitochondrial. The nuclear genome is the largest and typically encodes in excess of 27,000 genes using from 130 to several thousand megabases depending on plant species. In contrast, the chloroplast genome is typically made up of just 150 thousand base pairs into which 120 genes are tightly packed. However, a leaf cell can contain in excess of 100 chloroplasts each with 100 or more copies of the chloroplast genome. Thus, despite representing less than 0.1% of the sequence complexity of the cell, chloroplasts can contribute over 10% of the DNA content. In part because of this, genes located on the chloroplast genome can produce much higher levels of protein than an equivalent single copy gene located on the nuclear genome (up to 300 fold higher). In addition, chloroplasts are excluded from pollen and the chloroplast DNA is only inherited from the pollinated and not the pollinating crop plant. This has made chloroplasts very attractive as "green factories" for producing novel high value proteins, metabolites and bio-polymers, where high levels of gene expression are required. In addition, research groups are interested in improving photosynthetic efficiency by manipulating key proteins such as re-engineering the CO2 fixing RuBisCO large subunit or replacing it with that from plants or algae adapted to different light environments. However, such experiments are currently beyond the capabilities of the existing plastid engineering technology. Chloroplasts encode the ribosomal and tRNAs necessary for supporting the protein synthesis of the coding sequences present on their genome (mostly related to housekeeping functions and photosynthesis), but the majority of proteins found in the chloroplast (including aminoacyl-tRNA synthetases and RNA pol subunits) are encoded on the nuclear genome and imported from the cytoplasm.Direct transformation of the chloroplast genome was first achieved thirty years ago, but remains technically challenging due to the difficulty in introducing large DNA elements and the problem of ensuring transgenic plastid genomes fully replace the unmodified ones. Similar problems would also exist if attempts to replace large sections of the genome with fully synthetic sequence were to be made. We have recently addressed two of these problems using a two-component gene drive system and plants engineered to contain a single giant chloroplast instead of >100 smaller ones. Utilising this system, we will construct and test a plastid engineering tool set for 1) the iterative introduction of large cassettes for the introduction of complex biochemical pathways and 2) for carrying out targeted genome rearrangements, ultimately allowing substantial sections of the plastid genome to be replaced with bespoke synthetic versions.