Sengupta Annesha, Bandyopadhyay Anindita, Sarkar Debolina, Hendry John I, Schubert Max G, Liu Deng, Church George M, Maranas Costas D, Pakrasi Himadri B
Department of Biology, Washington University, St. Louis, Missouri, USA.
Department of Chemical Engineering, Pennsylvania State University, State College, Pennsylvania, USA.
mBio. 2024 Mar 13;15(3):e0353023. doi: 10.1128/mbio.03530-23. Epub 2024 Feb 15.
Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here, we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR-Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55-kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the wild type (WT). This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome-to-phenome relationships.IMPORTANCEGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.
蓝藻是光合生物，作为可持续生物生产的潜在宿主已获得广泛认可。然而，它们复杂的调控网络给主要的代谢工程努力带来了重大挑战，从而限制了它们作为生产宿主的可行性。基因组精简已被证明是提高异养生物生产力和适应性的一种成功方法，但在光合生物中尚未充分发挥其潜力。在此，我们展示了对生长最快的蓝藻UTEX 2973基因组的系统性缩减。这项工作是光合自养生物中的首例，涉及一个迭代过程，该过程使用了由实验分析和计算工具指导的最先进的基因组编辑技术。CRISPR-Cas3实现了对预测的可 dispensable 区域的大规模、逐步删除，并有助于鉴定必需基因。将这些大的删除区域合并，获得了一个基因组减少55 kb的菌株。与野生型（WT）相比，基因组精简的菌株在生长（提高了23%）和生产力（提高了22.7%）方面表现出改善。这种精简策略不仅有可能培育出生长和生产力性状得到改善的蓝藻菌株，还能促进对其基因组与表型关系的更好理解。
重要性
基因组精简是自然生命系统采用的一种进化策略，用于从其基因组中去除不必要的基因，作为一种适应和进化的机制。虽然这种策略已成功应用于开发具有所需表型的合成异养微生物系统，但在光合自养生物中尚未得到广泛探索。基因组精简策略结合了计算预测以识别可 dispensable 区域，并使用基因组编辑工具进行实验验证，在本研究中，我们采用了一种改进策略，目标是将基因组大小最小化到在特定条件下允许最佳细胞适应性的程度。我们的策略在光合自养生物中探索了一种新型基因组编辑工具，与其他现有工具不同，它能够从基因组中进行大规模、自发的最佳删除。我们的研究结果证明了这种改进策略在获得基因组精简、适应性和生产力提高的菌株方面具有有效性。
