Lu Jiangong, Wu Yaokang, Deng Chen, Liu Yanfeng, Lv Xueqin, Li Jianghua, Du Guocheng, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China.
Biotechnol Notes. 2022 Feb 5;3:15-24. doi: 10.1016/j.biotno.2022.02.001. eCollection 2022.
N-acetylglucosamine (GlcNAc), a glucosamine derivative, has a wide range of applications in pharmaceutical fields, and there is an increasing interest in the efficient production of GlcNAc genetic engineered bacteria. In this work, ATCC 25947 (DE3) strain was engineered by a model-based dynamic regulation strategy achieving GlcNAc overproduction. First, the GlcNAc synthetic pathway was introduced into , and through flux balance analysis of the genome-scale metabolic network model, metabolic engineering strategies were generated to further increase GlcNAc yield. Knock-out of genes and encoding pyruvate oxidase and lactate dehydrogenase, increased GlcNAc titer by 5.1%. Furthermore, knocking out N-acetylmuramic acid 6-phosphate etherase encoded by and enhancing glutamine synthetase encoded by gene further increased GlcNAc titer to 130.8 g/L. Analysis of metabolic flux balance showed that GlcNAc production maximization requires the strict dynamic restriction of the reactions catalyzed by and to balance cell growth and product synthesis. Hence, a dynamic regulatory system was constructed by combining the CRISPRi (clustered regularly interspaced short palindromic repeats interference) system with the lactose operon and the transcription factor pdhR, allowing the cell to respond to the concentration of pyruvate and IPTG to dynamically repress and transcription. Finally, the engineered bacteria with the dynamic regulatory system produced 143.8 g/L GlcNAc in a 30-L bioreactor in 55 h with a yield reaching 0.539 g/g glucose. Taken together, this work significantly enhanced the GlcNAc production of Moreover, it provides a systematic, effective, and universal way to improve the synthetic ability of other engineered strains.
N-乙酰葡糖胺（GlcNAc）是一种葡糖胺衍生物，在制药领域有广泛应用，人们对利用基因工程菌高效生产GlcNAc的兴趣与日俱增。在本研究中，通过基于模型的动态调控策略对ATCC 25947（DE3）菌株进行工程改造以实现GlcNAc的过量生产。首先，将GlcNAc合成途径引入该菌株，并通过对基因组规模代谢网络模型进行通量平衡分析，制定代谢工程策略以进一步提高GlcNAc产量。敲除编码丙酮酸氧化酶和乳酸脱氢酶的基因 和 ，使GlcNAc滴度提高了5.1%。此外，敲除 编码的N-乙酰胞壁酸6-磷酸醚酶并增强 基因编码的谷氨酰胺合成酶，使GlcNAc滴度进一步提高至130.8 g/L。代谢通量平衡分析表明，GlcNAc产量最大化需要严格动态限制由 和 催化的反应，以平衡细胞生长和产物合成。因此，通过将CRISPRi（成簇规律间隔短回文重复序列干扰）系统与乳糖操纵子 以及转录因子pdhR相结合，构建了一个动态调控系统，使细胞能够响应丙酮酸和IPTG的浓度来动态抑制 和 的转录。最终，具有动态调控系统的工程菌在30 L生物反应器中于55 h内生产了143.8 g/L的GlcNAc，产率达到0.539 g/g葡萄糖。综上所述，本研究显著提高了GlcNAc的产量。此外，它为提高其他工程菌株的合成能力提供了一种系统、有效且通用的方法。
