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Random Base Editing for Genome Evolution in Saccharomyces cerevisiae
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2021-09-20 , DOI: 10.1021/acssynbio.1c00217
Yingjia Pan 1 , Siyang Xia 1 , Chang Dong 1, 2 , Haojie Pan 1 , Jin Cai 1 , Lei Huang 1, 2 , Zhinan Xu 1 , Jiazhang Lian 1, 2
Affiliation  

Because of the limited understanding of cellular metabolism and regulatory networks, the rational engineering of complex industrial traits remains a grand challenge for the construction of microbial cell factories. Thus the development of simple, efficient, and programmable genome evolution techniques is still in high demanded for industrial biotechnology. In the present study, we established a random base editing (rBE) system for genome evolution in Saccharomyces cerevisiae. By fusing an unspecific single-stranded DNA (ssDNA)-binding protein to a cytidine deaminase, rBE introduced C to T mutations in a genome-wide manner. Specifically, we chose DNA-replication-related proteins, including replication factor A (RFA1, RFA2, and RFA3), DNA primase (PRI1), DNA helicase A (HCS1), and topoisomerase I (TOP1), to mediate the deamination of genomic ssDNA. As a proof of concept, we roughly estimated the rBE-mediated yeast genome mutation rate using the CAN1 mutation/canavanine resistance reporter system. We then evaluated the performance of these rBEs in improving the resistance against isobutanol and acetate and increasing the production of β-carotene. Finally, we employed the optimal rBE for the continuous genome evolution of a yeast cell factory resistant to 9% isobutanol. Owing to the conservation of DNA replication mechanisms, rBE is generally applicable and theoretically can be adopted for the continuous genome evolution of all organisms.

中文翻译:

酿酒酵母基因组进化的随机碱基编辑

由于对细胞代谢和调控网络的了解有限,复杂工业性状的合理工程仍然是微生物细胞工厂建设的巨大挑战。因此,工业生物技术仍然迫切需要开发简单、高效和可编程的基因组进化技术。在本研究中,我们建立了用于酿酒酵母基因组进化的随机碱基编辑 (rBE) 系统. 通过将非特异性单链 DNA (ssDNA) 结合蛋白与胞苷脱氨酶融合,rBE 以全基因组方式引入了 C 到 T 突变。具体来说,我们选择了 DNA 复制相关蛋白,包括复制因子 A(RFA1、RFA2 和 RFA3)、DNA 引发酶(PRI1)、DNA 解旋酶 A(HCS1)和拓扑异构酶 I(TOP1),以介导基因组的脱氨基作用。单链DNA。作为概念证明,我们使用CAN1粗略估计了 rBE 介导的酵母基因组突变率突变/刀豆氨酸抗性报告系统。然后,我们评估了这些 rBE 在提高对异丁醇和乙酸盐的耐受性以及增加 β-胡萝卜素产量方面的性能。最后,我们采用了最佳的 rBE 来实现对 9% 异丁醇具有抗性的酵母细胞工厂的持续基因组进化。由于DNA复制机制的保守性,rBE具有普遍适用性,理论上可用于所有生物的基因组持续进化。
更新日期:2021-10-15
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