Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose

Anthranilate and its derivative aniline are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, aniline is mainly chemically produced from petroleum-derived benzene, but it could be also obtained more sustainably by decarboxylation of the microbially produced shikimate pathway intermediate anthranilate. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy- arabinoheptulosonate-7-phosphate synthase from E. coli, catalyzing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based in vivo screening strategy for identifying variants with increased feedback-resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production, but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates, or any other aromatic compound derived thereof.