Enhancing glutamine production by optimising the GS-GOGAT pathway in Corynebacterium glutamicum and NH 4 + -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 4 + -limited fermentation process limits the concentration of NH 4 + 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 4 + -limited fermentation process, the effects of starting feeding with (NH 4 ) 2 SO 4 at different times of fermentation and three (NH 4 ) 2 SO 4 feeding strategies on glutamine fermentation were studied, and a NH 4 + -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 4 + -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%.