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Synergistic improvement of N-acetylglucosamine production by engineering transcription factors and balancing redox cofactors
Metabolic Engineering ( IF 6.8 ) Pub Date : 2021-07-28 , DOI: 10.1016/j.ymben.2021.07.012
Chen Deng 1 , Xueqin Lv 1 , Jianghua Li 2 , Hongzhi Zhang 3 , Yanfeng Liu 1 , Guocheng Du 4 , Rodrigo Ledesma Amaro 5 , Long Liu 1
Affiliation  

The regulation of single gene transcription level in the metabolic pathway is often failed to significantly improve the titer of the target product, and even leads to the imbalance of carbon/nitrogen metabolic network and cofactor network. Global transcription machinery engineering (gTME) can activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways, so transcription factors with specific functions can be expressed according to different metabolic regulation requirements, thus effectively increasing the synthesis of target metabolites. In addition, maintaining intracellular redox balance through cofactor engineering can realize the self-balance of cofactors and promote the efficient synthesis of target products. In this study, we rebalanced the central carbon/nitrogen metabolism and redox metabolism of Corynebacterium glutamicum S9114 by gTME and redox cofactors engineering to promote the production of the nutraceutical N-acetylglucosamine (GlcNAc). Firstly, it was found that the overexpression of the transcription factor RamA can promote GlcNAc synthesis, and the titer was further improved to 16 g/L in shake flask by using a mutant RamA (RamAM). Secondly, a CRISPR interference (CRISPRi) system based on dCpf1 was developed and used to inhibit the expression of global negative transcriptional regulators of GlcNAc synthesis, which promoted the GlcNAc titer to 27.5 g/L. Thirdly, the cofactor specificity of the key enzymes in GlcNAc synthesis pathway was changed by rational protein engineering, and the titer of GlcNAc in shake flask was increased to 36.9 g/L. Finally, the production of GlcNAc was scaled up in a 50-L fermentor, and the titer reached 117.1 ± 1.9 g/L, which was 6.62 times that of the control group (17.7 ± 0.4 g/L), and the yield was increased from 0.19 g/g to 0.31 g/g glucose. The results obtained here highlight the importance of engineering the global regulation of central carbon/nitrogen metabolism and redox metabolism to improve the production performance of microbial cell factories.



中文翻译:

通过改造转录因子和平衡氧化还原辅因子协同改进 N-乙酰氨基葡萄糖的生产

代谢途径中单基因转录水平的调控往往不能显着提高目标产物的效价,甚至导致碳/氮代谢网络和辅因子网络的失衡。全局转录机械工程(gTME)可以激活或抑制特定代谢途径中多个基因的协同表达,从而可以根据不同的代谢调控要求表达具有特定功能的转录因子,从而有效增加目标代谢物的合成。此外,通过辅因子工程维持细胞内氧化还原平衡,可以实现辅因子的自我平衡,促进目标产物的高效合成。在这项研究中,我们重新平衡了中央碳/氮代谢和氧化还原代谢谷氨酸棒杆菌S9114 通过 gTME 和氧化还原辅因子工程促进营养保健品 N-乙酰氨基葡萄糖 (GlcNAc) 的产生。首先发现转录因子RamA的过表达可以促进GlcNAc的合成,通过使用突变体RamA(RamAM),在摇瓶中的效价进一步提高到16 g/L。其次,开发了基于dCpf1的CRISPR干扰(CRISPRi)系统,用于抑制GlcNAc合成的全局负转录调节因子的表达,促进GlcNAc滴度达到27.5 g/L。第三,通过合理的蛋白质工程改变了GlcNAc合成途径关键酶的辅因子特异性,摇瓶中GlcNAc的滴度提高到36.9 g/L。最后,GlcNAc在50-L发酵罐中按比例生产,滴度达到117.1±1.9g/L,是对照组(17.7±0.4 g/L)的6.62倍,产率从0.19 g/g提高到0.31 g/g葡萄糖。此处获得的结果强调了对中央碳/氮代谢和氧化还原代谢进行全局调节以提高微生物细胞工厂生产性能的重要性。

更新日期:2021-07-30
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