Fig. 3.

Pathway compartmentalization in mitochondria and peroxisomes of S. cerevisiae for mandelic acid production. A) and B) Intracellular localization of _mitAro7^fbr-sfGFP and sfGFP-_peroxAro7^fbr, respectively, visualized by fluorescence microscopy. CEN.PK2-1C cells expressing MTS-ARO7^fbr-sfGFP (A) and sfGFP-ARO7^fbr-ePTS1 (B) from plasmids were grown until the exponential phase in lf-SCD medium. The scale bars indicate 2 µm. C) and D) mandelic acid (MA, top) and hydroxymandelic acid (HMA, bottom) titers after 24 h of fermentation with MRY25 (CEN.PK2-1C TRP1 Shik↑ aro7Δ trp2Δ) with Aro7^fbr, Pha2 and N. uniformis HmaS targeted to mitochondria (M, red), cytosol (C, blue) or peroxisomes (P, green). Plain-colored bars present samples in which all three consecutive enzymes were localized in one compartment (blue, cytosol; red, mitochondria; green, peroxisomes). The fermentation was performed in SMD (20 g/L glucose) with anthranilate supplementation and a starting OD₆₀₀ of 5. Error bars indicate standard deviation of biological duplicates (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).