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%.
Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum
