'21ENGBIO' Towards SYnthetic CHLOroPlastS (SYCHLOPS)

“21ENGBIO”迈向合成叶绿体 (SYCHLOPS)

• 批准号: BB/W01260X/1
• 负责人: Katalin Kovacs
• 金额: $ 12.82万
• 依托单位: University of Nottingham
• 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: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW01260X%2F1
• 关键词: 21ENGBIO; Towards; SYnthetic; CHLOroPlastS; SYCHLOPS

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.

具有完全或部分合成基因组的细菌和酵母已经被创造出来，这些被证明是工程生物学的有用平台。例如，一个设计良好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，以允许扩展遗传密码。一个很好的例子是大肠杆菌菌株Syn61，其中所有的丝氨酸UCG和UCA密码子与所有UAG终止密码子一起被替换。这使得这些密码子可以重新分配，并将两种非规范的氨基酸加入到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，创建完整或部分合成的基因组具有挑战性、耗时和成本。植物包含三个基因组，即核、叶绿体(叶绿体)和线粒体。核基因组是最大的，通常编码超过27,000个基因，根据植物物种的不同，使用130到数千个兆基。相比之下，叶绿体基因组通常只由15万个碱基对组成，其中有120个基因紧密结合在一起。然而，一个叶细胞可以包含超过100个叶绿体，每个叶绿体有100个或更多的叶绿体基因组拷贝。因此，尽管不到细胞序列复杂性的0.1%，但叶绿体可以贡献超过10%的DNA含量。部分原因是，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同等单一拷贝基因(高达300倍)高得多的蛋白质水平。此外，叶绿体被排除在花粉之外，叶绿体DNA只从授粉的作物中遗传，而不是从授粉的作物中遗传。这使得叶绿体成为生产新的高价值蛋白质、代谢物和生物聚合物的“绿色工厂”，在这些地方需要高水平的基因表达。此外，研究小组对通过操纵关键蛋白质来提高光合作用效率感兴趣，例如重新设计固定Rubisco大亚基的二氧化碳，或者用适应不同光环境的植物或藻类的二氧化碳亚基取代它。然而，这样的实验目前超出了现有的叶绿体工程技术的能力。叶绿体编码核糖体和tRNA，以支持其基因组上编码序列的蛋白质合成(主要与管家功能和光合作用有关)，但叶绿体中的大多数蛋白质(包括氨基酰-tRNA合成酶和RNA Poll亚基)是在核基因组上编码的，并从细胞质中输入。叶绿体基因组的直接转化在30年前首次实现，但由于引入大DNA元件的困难和确保转基因叶绿体基因组完全取代未修饰的叶绿体基因组的问题，在技术上仍然具有挑战性。如果试图用完全合成的序列取代大段基因组，也会存在类似的问题。最近，我们使用双组分基因驱动系统和含有单个巨大叶绿体而不是更小的叶绿体的植物，解决了其中的两个问题。利用这一系统，我们将构建和测试一套叶绿体工程工具集，用于1)迭代引入大型盒式磁带，用于引入复杂的生化途径，2)进行定向基因组重排，最终允许用定制的合成版本取代叶绿体基因组的大部分片段。