PDF(Pubmed)
Abstract：
High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the \'dock\' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.
摘要：
高的自发突变率对于获得理想的表型和探索基因与表型之间的关系至关重要。如何打破生物体的遗传稳定性,提高突变频率成为研究热点。这里,我们提出了一种实用且可控的进化工具（oMut-Cgts），该工具基于谷氨酸棒杆菌的双遗传水平修饰工程。首先,基于RNA聚合酶α亚基和DNA解旋酶Cgl0854作为胞苷脱氨酶（pmCDA1）的“码头”的转录和复制水平的修饰工程显着增加了突变率，证明pmCDA1在瞬时ssDNA周围的定位是基因组突变所必需的。然后，双遗传水平的联合改造和工程优化使突变率提高了1.02×104倍。基因组测序表明，oMut-Cgts在全基因组范围内执行均匀有效的C：G→T：A转换。此外，oMut-Cgts介导的谷氨酸棒杆菌在胁迫下的快速进化(酸，氧化和乙醇）耐受性证明该工具在多维生物工程（快速表型进化，基因功能挖掘和蛋白质进化)。本研究中提供的快速基因组进化策略有望适用于所有原核细胞的各种应用。
