Synergetic engineering of central carbon and nitrogen metabolism for the production of N-acetylglucosamine in Bacillus subtilis
Synergetic engineering of central carbon and nitrogen metabolism for the production of N-acetylglucosamine in Bacillus subtilis
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枯草芽孢杆菌生产 N-乙酰氨基葡萄糖的中心碳氮代谢协同工程
DOI：
10.1002/bab.1845
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发表时间：
2019-12-29
影响因子：
2.8
通讯作者：
Liu, Long
中科院分区：
文献类型：
--
作者：
Niu, Tengfei;Lv, Xueqin;Liu, Long
N-乙酰葡萄糖（GLCNAC）是一种含氮化合物，被广泛用作营养和药物。在以前的工作中，我们通过工程化中心碳代谢来构建了一种重组枯草芽孢杆菌菌株，用于GLCNAC的生物合成。但是，氮的合成也需要氮。因此，有必要同时协调碳和氮代谢以改善GlcNAC的产生。在这项工作中，我们试图通过增加前体N-乙酰基葡萄糖6-磷酸（GLCNAC6P）和谷氨酸的供应来增强枯草芽孢杆菌的GlcNAC产生。通过工程化启动子和核糖体结合位点来增强键酶，GlcNAC6P N-乙酰基转移酶（GNA1）的表达，从而增强GlcNAC6P的产生。接下来，我们检查了不同氮源对GlcNAC合成的影响。我们观察到尿素可以促进GlcNAC合成的氮同化。通过删除两个内源性谷氨酸脱氢酶基因（ROCG和GUDB），并整合一个外源性谷氨酸脱氢酶基因（GDH），从而改善了谷氨酸合成。该策略分别将细胞内谷氨酸和谷氨酰胺分别提高了69.8％和46.9％。中央碳和氮代谢的协同工程将GLCNAC滴度从14.0增加到22.2 g/L。因此，我们的研究证明了碳和氮代谢协调在含氮化合物的产生中的重要性。
N-acetylglucosamine (GlcNAc) is a nitrogen-containing compound, which is widely used as a nutraceutical and pharmaceutical. In our previous work, we constructed a recombinant Bacillus subtilis strain for the biosynthesis of GlcNAc by engineering the central carbon metabolism. However, nitrogen is also required for the synthesis of GlcNAc. Hence, it is necessary to simultaneously coordinate the carbon and nitrogen metabolism to improve production of GlcNAc. In this work, we attempted to enhance GlcNAc production in B. subtilis by increasing supply of precursors N-acetylglucosamine 6-phosphate (GlcNAc6P) and glutamate. The expression of a key enzyme, GlcNAc6P N-acetyltransferase (GNA1), was enhanced by engineering the promoter and ribosome binding site to enhance the production of GlcNAc6P. Next, we examined the effect of different nitrogen sources on GlcNAc synthesis. We observed that urea can promote nitrogen assimilation for GlcNAc synthesis. The glutamate synthesis was improved by deleting the two endogenous glutamate dehydrogenase genes (rocG and gudB) and by integrating one exogenous glutamate dehydrogenase gene (gdh). This strategy enhanced the intracellular glutamate and glutamine by 69.8% and 46.9%, respectively. The synergetic engineering of central carbon and nitrogen metabolisms increased the GlcNAc titer from 14.0 to 22.2 g/L in the shaker flask. Hence, our study demonstrated the importance of carbon and nitrogen metabolism coordination in the production of nitrogen-containing compounds.