A Novel, Efficient Biotransformation for the Production of L–Phenylalanine

Abstract

We report the isolation of a Corynebacterium equi strain that produces more than 33 g/l of L–phenylalanine from α–acetamidocinnamic acid in molar yields greater than 99% using immobilised whole cells. The metabolic pathway, by which the cells carry out the conversion, contains an inducible permease, an acylase and a dehydrogenase. The achiral α–acetamidocinnamic acid is actively transported into the cells where it under goes deacetylation and deamination, via the acylase, to yield the stable intermediate, phenylpyruvic acid. An inducible phenylalanine dehydrogenase then catalyses the reductive amination of the phenylpyruvate to yield optically pure L–phenylalanine. The overall reaction was limited by the supply of NADH, but was successfully manipulated by the addition of oxidisable co–substrates, such as lactate, pyruvate and formate. Mutants constitutive for α–acetamidocinnamate conversion contain increased activities of both the acylase and phenylalanine dehydrogenase. One such mutant, OAR1–16, produced L–phenylalanine approximately 2.5 times faster than the parent, was insensitive to end product inhibition and accumulated more than 30 g/l L–phenylalanine. The use of a substrate feedstock containing α–acetamidocinnamate, lactate and NH₄OH permitted the continuous production of L–phenylalanine, at pH 7.0 and 34°C, coupling the lactate and phenylalanine dehydrogenases as a system for intra–cellular recycling of reducing equivalents. The commercial viability of this process is discussed.