Abstract
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 22.2g/L in the shaker flask. Hence, our study demonstrated the importance of carbon and nitrogen metabolism coordination in the production of nitrogen-containing compounds.
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