'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.

已经产生了具有完全或部分合成基因组的细菌和酵母菌，这些细菌和部分合成基因组已被证明是工程生物学的有用平台。例如，设计良好的合成基因组可以随意交换和重新排列关键基因。此外，已重新分配了同义密码子和UAG终止密码子，以允许扩展的遗传密码。一个很好的例证是大肠杆菌菌株Syn61，其中所有丝氨酸UCG和UCA密码子都与所有UAG终止密码子一起替换。该巡回赛允许对这些密码子进行重新分配，并将两种非规范氨基酸掺入由引入转基因制成的蛋白质中。但是，酵母和细菌基因组相对较大，创建完整或部分合成的版本具有挑战性，耗时且昂贵。植物包含三个基因组，核成形棒（质体）和线粒体。核基因组是最大的，通常使用130到数千兆巴的27,000个基因编码超过27,000个基因，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个基因被紧密地填充。但是，叶片细胞可以包含超过100个叶绿体，每个叶绿体具有100份或更多份叶绿体基因组。因此，尽管占细胞序列复杂性的0.1％，但叶绿体仍可贡献超过10％的DNA含量。在某种程度上，位于叶绿体基因组上的基因比位于核基因组上的等效单拷贝基因（高达300倍）的基因可以产生更高的蛋白质水平。此外，叶绿体被排除在花粉中，叶绿体DNA仅从授粉而不是授粉的农作物植物遗传。这使叶绿体具有非常吸引人的吸引力，就像“绿色工厂”，用于产生新型的高价值蛋白质，代谢产物和生物聚合物，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（例如重新设计co2固定rubisco大亚基）或用适合不同光环境的植物或藻类替换的二氧化碳来提高光合效率。但是，此类实验目前超出了现有的塑料工程技术的功能。叶绿体编码了支撑其基因组中存在的编码序列的蛋白质合成所必需的核糖体和TRNA（主要与管家功能和光合作用有关），但在叶绿体中发现的大多数蛋白质（包括氨基酰基囊酶和RNA pol subunits）在叶绿体中的大多数蛋白质是核基因的转换，并在核基因中均具有核基因和导入，并在核基因上进行了指示。叶绿体基因组最初是在30年前实现的，但由于难以引入大型DNA元素以及确保转基因质体基因组完全取代未修饰的基因组的问题，因此在技术上仍然具有挑战性。如果尝试用完全合成序列代替大型基因组的大部分部分，也将存在类似的问题。最近，我们使用两个组件基因驱动系统和工厂设计的植物解决了这些问题中的两个问题，这些植物含有单个巨型叶绿体，而不是> 100个较小的叶绿体。利用该系统，我们将构建和测试一个针对1）迭代引入大型盒式的塑料工程工具，用于引入复杂的生化途径，2）用于进行有针对性的基因组重排，最终允许质质体基因组的实质性部分被Bespoke基因组替换为Bespoke合成版本。