Efficient enzymatic production of benzaldehyde from l-phenylalanine with a mutant form of 4-hydroxymandelate synthase

Benzaldehyde is an organic compound with an almond-like aroma and one of the most important and widely used flavorings in the food industry. To develop an enzymatic process for the production of benzaldehyde from l-phenylalanine, four enzymes were expressed in Escherichia coli; l-amino acid deaminase, 4-hydroxymandelate synthase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase. Although each E. coli strain could be used to synthesize benzaldehyde from l-phenylalanine, the yield was low due to the accumulation of an intermediate, phenylpyruvic acid. We developed a second reaction step by engineering 4-hydroxymandelate synthase of Actinoplanes teichomyceticus. A quadruple mutant of 4-hydroxymandelate synthase (A199V/Q206R/I217V/K337Q) obtained by random and site-directed mutagenesis demonstrated 2.4-fold higher activity than wild type. Furthermore, the mutant-expressing strain was able to produce benzaldehyde from 100 mm l-phenylalanine at a conversion rate of 84% (wild type, 37%). We report the development of an efficient process for benzaldehyde production using l-phenylalanine as a substrate.