通过大规模基因组装和基因组整合进行的代谢工程
Metabolic Engineering of with Massive Gene Assembly and Genomic Integration.
作者信息
Cui Yuhui, Liu Duo, Xue Huimin, Li Mingshan, Guo Wenhong, Huang Cuiqin, Zheng Xintian, Yang Jichao, Liu Hong, Yin Huifang, Wang Hanjie
机构信息
School of Life Sciences, Faculty of Medicine, Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology, Tianjin Key Laboratory of Function and Application of Biological Macromolecular Structures, Tianjin University, Tianjin 300072, China.
State Key Laboratory of Synthetic Biology, Tianjin University, Tianjin 300072, China.
出版信息
ACS Synth Biol. 2025 Aug 15;14(8):3196-3206. doi: 10.1021/acssynbio.5c00333. Epub 2025 Jul 17.
Synthetic biology has developed varied strategies of one-pot multigene assembly and genomic integration to promote the genetic engineering of the chassis. However, such strategies for engineering oleaginous yeast is lacking, given the current stage that at most 5 exogenous genes (around 13 kb) can be assembled and integrated into the genome at once. Here, we developed a strategy of massive gene assembly and integration in the genome. As a proof of concept, dozen-gene assembly (more than 30 kb) and integration were achieved stably and reproducibly, and a chassis containing a total of 35 exogenous genes (a sum of 93.5 kb) was constructed. The introduction of massive genes modulated the synthesis of lycopene, a heterologous natural product, to quite different extents. Ultimately, an optimized constructed strain containing 15 exogenous genes achieved the highest yield of 144.58 mg/g DCW and produced a lycopene titer of 2144.83 mg/L in a 5 L bioreactor. Our strategy significantly expands the capability of genetic manipulation and metabolic engineering.
合成生物学已经开发出多种一锅多基因组装和基因组整合策略,以促进底盘细胞的基因工程。然而,鉴于目前最多只能一次性组装并整合5个外源基因(约13 kb)到基因组中的阶段,针对产油酵母的此类策略尚属缺乏。在此,我们开发了一种在基因组中进行大规模基因组装和整合的策略。作为概念验证,稳定且可重复地实现了十几个基因(超过30 kb)的组装和整合,并构建了一个总共包含35个外源基因(总计93.5 kb)的底盘细胞。大量基因的引入对番茄红素(一种异源天然产物)的合成产生了不同程度的调节。最终,一个含有15个外源基因的优化构建菌株在5 L生物反应器中实现了144.58 mg/g干细胞重的最高产量,番茄红素滴度达到2144.83 mg/L。我们的策略显著扩展了基因操作和代谢工程的能力。