Enhancing glutamine production by optimising the GS-GOGAT pathway in Corynebacterium glutamicum and NH4+-limited fermentation
Abstract: The GS-GOGAT pathway is a key metabolic pathway of glutamate and glutamine. Optimising this pathway, leading to metabolic flux to glutamine, can increase glutamine production and reduce the production of the by-product glutamate. The NH-limited fermentation process limits the concentration of NH to increase the activity of GS and further increase the yield of glutamine. The GS-GOGAT pathway was optimised by knocking out the GOGAT genes NCgl0181 and NCgl0182 and the glutaminase genes NCgl2395 and NCgl2500 and by integrating a copy of the GS gene glnAbsu from Bacillus subtilis and a copy of the glutamine synthase gene glnAlcb from Lactobacillus acidophilus into the genomic NCgl0182 and NCgl2500 sites. Furthermore, the pXT01 plasmid with the strong promoter tuf was used to overexpress glnAbsu and glnAlcb. To obtain an optimal NH-limited fermentation process, the effects of starting feeding with (NH)SO at different times of fermentation and three (NH)SO feeding strategies on glutamine fermentation were studied, and a NH-limited fermentation process that was the most suitable for glutamine fermentation was determined. After optimising the GS-GOGAT pathway, Corynebacterium glutamicum G-6 was subjected to the NH-limited fermentation process to greatly increase the production of glutamine. The yield of glutamine reached 98.7 g/L, which was 104.8% higher than that in the original strain GM34; the content of glutamate reached 4.5 g/L, which then decreased by 85.2%; the GS activity increased significantly, and the sugar-acid conversion rate reached 41.2%.