Zhang Yun, Shang Xiuling, Lai Shujuan, Zhang Yu, Hu Qitiao, Chai Xin, Wang Bo, Liu Shuwen, Wen Tingyi
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
University of Chinese Academy of Sciences , Beijing 100049, China.
ACS Synth Biol. 2018 Feb 16;7(2):635-646. doi: 10.1021/acssynbio.7b00373. Epub 2018 Jan 19.
l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived CH to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.
L-丝氨酸是DNA生物合成的主要一碳源，由于L-丝氨酸分解代谢与微生物生长的耦合，微生物很难积累L-丝氨酸。在此，我们对谷氨酸棒杆菌中的一碳单位代谢途径进行了重新编程，以使L-丝氨酸分解代谢与细胞生长解偶联。基于计算机模拟的模型显示，编码丝氨酸羟甲基转移酶的glyA通量对L-丝氨酸生产率有负面影响。glyA转录的减弱导致L-丝氨酸积累增加、嘌呤库减少、生长不良和细胞形态变长。将来自大肠杆菌的由gcvTHP编码的甘氨酸裂解（Gcv）系统引入谷氨酸棒杆菌，使甘氨酸衍生的CH被同化为细胞内嘌呤合成，这导致一碳单位数量增加。引入Gcv不仅恢复了细胞活力和形态，还增加了L-丝氨酸的积累。此外，比较蛋白质组学分析表明，参与一碳单位循环的酶的丰度变化可能负责维持一碳单位的稳态。一碳代谢途径的重新编程使细胞在基本培养基中通过分批补料发酵达到可比的生长速率，以积累13.21 g/L的L-丝氨酸。这种新策略通过引入外源途径，为细胞特性调控和必需代谢物积累提供了新的见解。
