Stepwise metabolic engineering of Corynebacterium glutamicum for the production of phenylalanine

Abstract

Corynebacterium glutamicum was metabolically engineered to produce phenylalanine, a valuable aromatic amino acid that can be used as a raw material in the food and pharmaceutical industries. First, a starting phenylalanine-producer was constructed by overexpressing tryptophan-sensitive 3-deoxy-d-arabino-heptulosonate-7-phosphate syn-thase and phenylalanine-and tyrosine-insensitive bifunctional enzyme chorismate mutase-prephenate dehydratase from Escherichia coli, followed by the inactivation of enzymes responsible for the formation of dihydroxyacetone and the consumption of shikimate pathway-related compounds. Second, redirection of the carbon flow from tyrosine to phe-nylalanine was attempted by deleting of the tyrA gene encoding prephenate dehydrogenase, which catalyzes the committed step for tyrosine biosyn-thesis from prephenate. However, suppressor mutants were generated, and two mutants were isolated and examined for phenylalanine production and genome sequencing. The suppressor mutant harboring an amino acid exchange (L180R) on RNase J, which was experimentally proven to lead to a loss of function of the enzyme, showed signifi-cantly enhanced production of phenylalanine. Finally, modifications of phosphoenolpyruvate-py-ruvate metabolism were investigated, revealing that the inactivation of either phosphoenolpyruvate carboxylase or pyruvate carboxylase, which are enzymes of the anaplerotic pathway, is an effective means for improving phenylalanine production. The resultant strain, harboring a phosphoenolpyruvate carboxylase deficiency, synthesized 50.7 mM phe-nylalanine from 444 mM glucose. These results not only provided new insights into the practical mutations in constructing a phenylalanine-producing C. glutamicum but also demonstrated the creation of a potential strain for the biosynthesis of phenyl-alanine-derived compounds represented by plant secondary metabolites.