Enhancing l-glutamine production in Corynebacterium glutamicum by rational metabolic engineering combined with a two-stage pH control strategy

l-glutamine is a semi-essential amino acid widely used in the food and pharmaceutical industries. The microbial synthesis of l-glutamine is limited by lack of effective strains with high titer and safety. First, ARTP mutagenesis combined with high-throughput screening generated an l-glutamine-producing strain of Corynebacterium glutamicum with titer of 25.7 ± 2.7 g/L. Subsequently, a series of rational metabolic approaches were used to further improve l-glutamine production, which included increasing the carbon flow to l-glutamine (proB and NCgl1221 knockout), enhancing the catalytic efficiency of the key enzyme (glnE knockout and glnA screening and overexpression) and reinforcement of ATP regeneration (ppk overexpression and RBS optimization). Finally, we proposed a two-stage pH control strategy to address the inconsistent effect of pH on cell growth and l-glutamine production. These combined strategies led to a 186.0% increase of l-glutamine titer compared to that of the initial strain, reaching 73.5 ± 3.1 g/L with a yield of 0.368 ± 0.034 g/g glucose.