Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, People's Republic of China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, People's Republic of China.
Biotechnol Appl Biochem. 2020 Jan;67(1):123-132. doi: 10.1002/bab.1845. Epub 2019 Dec 29.
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.
N-乙酰葡萄糖胺（GlcNAc）是一种含氮化合物，广泛用作营养保健品和药物。在我们之前的工作中，通过工程化中心碳代谢，构建了一株用于 GlcNAc 生物合成的重组枯草芽孢杆菌菌株。然而，GlcNAc 的合成也需要氮。因此，有必要同时协调碳氮代谢以提高 GlcNAc 的产量。在这项工作中，我们试图通过增加 GlcNAc6P 和谷氨酸前体的供应来提高枯草芽孢杆菌中 GlcNAc 的产量。通过工程化启动子和核糖体结合位点来增强 GlcNAc6P N-乙酰转移酶（GNA1）的表达，从而增强 GlcNAc6P 的产生。接下来，我们研究了不同氮源对 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），谷氨酸的合成得到了改善。该策略分别将细胞内谷氨酸和谷氨酰胺提高了 69.8%和 46.9%。中心碳氮代谢的协同工程使摇瓶中的 GlcNAc 产量从 14.0 克/升增加到 22.2 克/升。因此，我们的研究表明了在含氮化合物生产中碳氮代谢协调的重要性。
