Bacterial bifunctional chorismate mutase-prephenate dehydratase PheA increases flux into the yeast phenylalanine pathway and improves mandelic acid production.

Mandelic acid is an important aromatic fine chemical and is currently mainly produced via chemical synthesis. Recently, mandelic acid production was achieved by microbial fermentations using engineered and expressing heterologous hydroxymandelate synthases (). The best-performing strains carried a deletion of the gene encoding the first enzyme of the tyrosine biosynthetic pathway and therefore were auxotrophic for tyrosine. This was necessary to avoid formation of the competing intermediate hydroxyphenylpyruvate, the preferred substrate for HmaS, which would have resulted in the predominant production of hydroxymandelic acid. However, feeding tyrosine to the medium would increase fermentation costs. In order to engineer a tyrosine prototrophic mandelic acid-producing strain, we tested three strategies: (1) rational engineering of the HmaS active site for reduced binding of hydroxyphenylpyruvate, (2) compartmentalization of the mandelic acid biosynthesis pathway by relocating HmaS together with the two upstream enzymes chorismate mutase Aro7 and prephenate dehydratase Pha2 into mitochondria or peroxisomes, and (3) utilizing a feedback-resistant version of the bifunctional enzyme PheA (PheA) in an deletion strain. PheA has both chorismate mutase and prephenate dehydratase activity. Whereas the enzyme engineering approaches were only successful in respect to reducing the preference of HmaS for hydroxyphenylpyruvate but not in increasing mandelic acid titers, we could show that strategies (2) and (3) significantly reduced hydroxymandelic acid production in favor of increased mandelic acid production, without causing tyrosine auxotrophy. Using the bifunctional enzyme PheA turned out to be the most promising strategy, and mandelic acid production could be increased 12-fold, yielding titers up to 120 mg/L. Moreover, our results indicate that utilizing PheA also shows promise for other industrial applications with that depend on a strong flux into the phenylalanine biosynthetic pathway.