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

Abstract

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum‐derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. 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 Escherichia coli, catalysing 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 a 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.

A New Direction for the Green, Environmentally Friendly and Sustainable Bioproduction of Aminobenzoic Acid and Its Derivatives

“…The enzyme anthranilate synthase (TrpEG), encoded by the trpEG gene, catalyzes the conversion of CHO into OABA and pyruvate, utilizing glutamine (Gln) as the amino donor. OABA serves as the first intermediate in the tryptophan biosynthesis pathway [25]. In addition to its role as anthranilate synthase, TrpD can also function as anthranilate phosphoribosyltransferase.…”

“…DAHP is converted into the intermediate shikimate through the conversion of 3-dehydroquinate (DHQ) and 3-dehydroshikimate (DHS). Subsequently, under the hydrolysis of ATP and the introduction of a second PEP, shikimate passes through two intermediates, shikimate-3-phosphate (S3P) and 5-enolpyruvyl-shikimate-3-phosphate (EPSP), and is converted into a common precursor CHO of aromatic amino acids [24,25].…”

“…In addition to E. coli, researchers have also studied the synthesis of OABA in other strains such as Pseudomonas putida, Saccharomyces cerevisiae, and Corynebacterium glutamicum. A modified C. glutamicum [25] strain using a mixture of glucose and xylose as a carbon source achieved a titer of 5.9 g/L in CGXII medium. However, the yields of the modified Pseudomonas putida [8] and Saccharomyces cerevisiae [27] were relatively lower, at 567.9 mg/L and 1.54 g/L, respectively.…”