Advancing the cyanobacterial cell factory: Synechocystis sp. 6803

蓝藻是光自养的原核生物，能够通过含氧光合作用将光转化为化学能。这种化学能通常用于促进生长和后代的产生，然而，已经证明，它可以从生长中重新定向，并以可再生的方式用于产生有用的碳基化合物，如燃料和药物。Synechocystis sp. 6803已成为该领域的蓝藻模型，因为它是单细胞的，自然可转化的，并且具有完全测序和注释的基因组。虽然在聚胞菌中已经成功地生产了多种碳基化合物，但其产量明显低于大肠杆菌或酿酒酵母。设计特定菌株、大规模种植、收获生物质和提取感兴趣产品的投入成本大大超过了所需产品的价值。缺乏对控制和调节基因表达的基本细胞过程的理解是造成这些低产量的原因。本项目旨在更详细地揭示Synechocystis sp. 6803的基因组结构、DNA复制机制和基因组拷贝数调控。现在人们很清楚，聚胞虫是多倍体，每个细胞的基因组拷贝的确切数量取决于生长阶段和环境条件。然而，目前尚不清楚基因组拷贝数波动的原因以及这一过程是如何调节的。此外，胞囊藻的染色体有一个尚未描述的复制起源，该领域的一些研究表明，原核生物普遍存在的DNA复制机制在胞囊藻中并不必要。最后，假设原核生物中的DNA自由地位于细胞质内，基因组组织是只有真核生物才有的现象；这个假设还有待证实。单纯组成性表达异源代谢途径只会导致遗传不稳定和宿主细胞的代谢负担。控制外源基因的表达是实现高产量的关键。通过揭示聚囊藻调节其基因组拷贝数、启动DNA复制和组织其基因组的分子过程，本研究希望为该领域的工作人员提供更好的控制异源基因在聚囊藻中表达的方式和时间，从而改进蓝藻细胞工厂的概念。

Cyanobacteria are photoautotrophic prokaryotes capable of converting light into chemical energy via oxygenic photosynthesis. This chemical energy is conventionally used to fuel growth and the generation of progeny however, it has been demonstrated that it can be redirected away from growth and used to generate useful carbon-based compounds such as fuels and pharmaceuticals in a renewable manner. Synechocystis sp. 6803 has become a model cyanobacterium in this field as it is unicellular, naturally transformable and possess a fully sequenced and annotated genome. Whilst a variety of carbon based compounds have been successfully produced in Synechocystis, the yields are significantly less than those achieved in Escherichia coli or Saccharomyces cerevisiae. The input costs of engineering specific strains, growing them at scale, harvesting biomass and extracting the product of interest significantly outweigh the value of the desired product. A lack of understanding of the basic cellular process that control and regulate gene expression is responsible for these low yields.This project aims to reveal a more detailed insight into the genome architecture, mechanisms of DNA replication and the regulation of genome copy number in Synechocystis sp. 6803. It is now well understood that Synechocystis is polyploid and the exact number of genome copies per cell fluctuates depending on the growth phase and environmental conditions. However, it is as of yet unknown why the genome copy number fluctuates and how this process is regulated. Furthermore, the chromosome of Synechocystis has an as of yet undescribed origin of replication and some studies in this area have suggested that the DNA replication machinery thought to be ubiquitous to prokaryotes is not essential in Synechocystis. And finally, it is assumed that DNA in prokaryotes is located freely within the cytosol and that genome organisation is a phenomenon reserved only for eukaryotes; an assumption that has yet to be confirmed. Simply constitutively expressing heterologous metabolic pathways has only resulted in genetic instability and a metabolic burden on the host cell. Control over heterologous gene expression is key to achieving high yields of desired products. By revealing the molecular processes by which Synechocystis regulates its genome copy number, initiates DNA replication and organises its genome, this study hopes to provide those working in this field with greater control over how and when heterologous genes are expressed in Synechocystis, thereby improving the notion of a cyanobacterial cell factory.