Robust production of N-acetyl-glucosamine in engineered Escherichia coli from glycerol-glucose mixture

Achieving a balanced cell growth and biosynthesis of target products is a pivotal challenge confronted in the realm of green biomanufacturing of chemicals, and it holds the promise of significantly enhancing the titer/yield/productivity of the target products. The co-utilization of carbon sources has emerged as a proven approach for the precise regulation of cell growth and biosynthesis processes, thereby serving as a potent strategy to expedite the production of chemicals. In our previous study, we successfully demonstrated the efficient production of N-acetyl-glucosamine in engineered Escherichia coli by leveraging an appropriate catabolic division of labor, utilizing a mixture of glycerol and glucose as the carbon source. In this study, we further refined the division of labor between these two carbon sources by meticulously regulating the expression of the zwf gene, which encodes glucose-6-phosphate dehydrogenase and is crucial in diverting carbon source to the pentose phosphate pathway (PPP). After comparing three strategies for balancing cell growth and production, the engineered strain NAG-1 with zwf knocked out aided by the optimization of the glycerol-to-glucose ratio and the feeding mode of carbon sources resulted in robust production of N-acetyl-glucosamine. Remarkable production in a 5 L bioreactor was achieved, obtaining 249 g/L of N-acetyl-glucosamine with a yield of 0.684 g/g of total carbon sources (specifically, 0.791 g/g of glucose) and a productivity of 3.46 g/L/h. These results establish NAG-1 as the most efficient microbial cell factory reported thus far for the bioproduction of N-acetyl-glucosamine. The robust production of N-acetyl-glucosamine in E. coli using a mixture of glycerol and glucose suggests the immense potential of mixed carbon sources in the industrial green biomanufacturing of chemicals.