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

Abstract

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 Escherichia coli and Saccharomyces cerevisiae expressing heterologous hydroxymandelate synthases (hmaS). 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 S. cerevisiae 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 E. coli enzyme PheA (PheAfbr) in an aro7 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 PheAfbr 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 PheAfbr also shows promise for other industrial applications with S. cerevisiae that depend on a strong flux into the phenylalanine biosynthetic pathway.

Endophytic Fungi of Native Salvia abrotanoides Plants Reveal High Taxonomic Diversity and Unique Profiles of Secondary Metabolites

“…Mandelic acid has been used as an antibacterial agent, particularly in the treatment of urinary tract infections (Putten, 1979). Recently, mandelic acid production was achieved by microbial fermentations using engineered Escherichia coli and Saccharomyces cerevisiae expressing heterologous hydroxymandelate synthases (Reifenrath et al, 2018). Moreover, caffeic acid was found in metabolite profiles of isolates assigned to the genera Talaromyces and Paraphoma.…”

Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products

“…We then decided to take what were now our best strains and see what the effect of modifications for increase of E4P and/or PEP would be. We also included a feedback resistant allele of KmARO3 since an ARO3 fbr created in S. cerevisiae was found to aid aromatic amino acid synthesis when used in tandem with ARO4 fbr [41]. Based on the performance of previous strains, we hypothesised that KmAro3p fbr would allow faster consumption of the increased E4P and PEP, avoiding their accumulation and diversion to other reactions.…”

Bioprocess Optimization for the Production of Aromatic Compounds With Metabolically Engineered Hosts: Recent Developments and Future Challenges

“…Another regulatory point is present at the chorismate branch, at which the chorismate mutase and prephenate dehydratase are feedback regulated by the end products, L-Phe and L-Try (Lütke-Eversloh and Stephanopoulos, 2005;Reifenrath et al, 2018). The most common strategies to overcome this bottleneck are the application of mutations that confer feedback resistance to chorismate mutase-prephenate dehydratase or the utilization of evolved genes (pheA ev ) (Báez-Viveros et al, 2004;Lütke-Eversloh and Stephanopoulos, 2005;Ikeda, 2006;Sprenger, 2007;Luttik et al, 2008).…”