Production of non-proteinogenic amino acids from α-keto acid precursors with recombinant Corynebacterium glutamicum

In the present work, Corynebacterium glutamicum was metabolically engineered for the enantioselective synthesis of non-proteinogenic amino acids as valuable building blocks for pharmaceuticals and agrochemicals. The novel bio-catalytic activity of C. glutamicum was obtained by heterologous expression of the branched chain aminotransferase IlvE from Escherichia coli. Upon this modification, the recombinant cells converted the α-keto acid precursor 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (HOAE) into the corresponding amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-amino ethanoic acid (HAAE). Similarly, also L-tert-leucine could be obtained from trimethyl pyruvate indicating a broader applicability of the novel strategy. In both cases, the amino group donor glutamate was supplied from the endogenous metabolism of the recombinant producer. Hereby, the uptake of the precursor and secretion of the product was supported by an enhanced cell permeability through treatment of ethambutol, which inhibits arabinosyl transferases involved in cell wall biosynthesis. The excretion of HAAE into the reaction medium was linked to the secretion of glutamate, indicating a similar mechanism for the export of both compounds. On the other hand, the efflux of L-tert-leucine appeared to be driven by active transport. Subsequent bioprocess engineering enabled HAAE and L-tert-leucine to be produced at a rate of 0.21 and 0.42mmol (gdry cells)^-1h^-1, respectively up to a final product titer of 40mM. Beyond the given examples, integrated metabolic and cell envelop engineering might extend the production of a variety of other non-proteinogenic amino acids as well as chiral amines by C. glutamicum.