Corynebacterium glutamicum as platform for the production of hydroxybenzoic acids

Nicolai Kallscheuer, Jan Marienhagen

Hydroxybenzoic acids are industrially relevant aromatic compounds, which also play key roles in the microbial carbon metabolism, e.g., as precursors for the synthesis of cofactors or metal-chelating molecules. Due to its pronounced resistance to aromatics Corynebacterium glutamicum represents an interesting platform for production of these compounds. Unfortunately, a complex catabolic network for aromatic molecules prevents application of C. glutamicum for microbial production of aromatic compounds other than aromatic amino acids, which cannot be metabolized by this microorganism. We completed the construction of the platform strain C. glutamicum DelAro5, in which the deletion of altogether 27 genes in five gene clusters abolished most of the peripheral and central catabolic pathways for aromatic compounds known in this microorganism. The obtained strain was subsequently applied for the production of 2-hydroxybenzoate (salicylate), 3-hydroxybenzoate, 4-hydroxybenzoate and protocatechuate, which all derive from intermediates of the aromatic amino acid-forming shikimate pathway. For an optimal connection of the designed hydroxybenzoate production pathways to the host metabolism, C. glutamicum was additionally engineered towards increased supply of the shikimate pathway substrates erythrose-4-phosphate and phosphoenolpyruvate by manipulation of the glucose transport and key enzymatic activities of the central carbon metabolism. With an optimized genetic background the constructed strains produced 0.01 g/L (0.07 mM) 2-hydroxybenzoate, 0.3 g/L (2.2 mM) 3-hydroxybenzoate, 2.0 g/L (13.0 mM) protocatechuate and 3.3 g/L (23.9 mM) 4-hydroxybenzoate in shaking flasks. By abolishing its natural catabolic network for aromatic compounds, C. glutamicum was turned into a versatile microbial platform for aromatics production, which could be exemplarily demonstrated by rapidly engineering this platform organism towards producing four biotechnologically interesting hydroxybenzoates. Production of these compounds was optimized following different metabolic engineering strategies leading to increased precursor availability. The constructed C. glutamicum strains are promising hosts for the production of hydroxybenzoates and other aromatic compounds at larger scales.