A case of TPA-tience: insights and roadblocks in terephthalic acid upcycling with Saccharomyces cerevisiae​

Yeasts are robust hosts for large-scale chemical fermentations, especially to produce organic acids, considering their ability to withstand their presence. In this work, we explored Saccharomyces cerevisiae as a microbial cell factory towards the upcycling of terephthalic acid (TPA), a primary monomer of polyethylene terephthalate, into protocatechuic acid (PCA), a chemical of interest for cosmetic formulations. While bacterial TPA catabolism is well-documented, research on yeast-based biotransformation remains scarce. Our findings show that wild-type S. cerevisiae is highly tolerant to TPA (up to 10 g L⁻¹), however deletion of the ABC transporter PDR12 increases sensitivity. This demonstrates Pdr12 involvement in TPA export and largely accounts for the intrinsic TPA resistance of yeast, with only limited passive membrane permeation. To enable the conversion of TPA to PCA, S. cerevisiae was engineered to express two different bacterial TPA dioxygenases (TPA-DO) and DCD dehydrogenases (DCD-DH), from either Ideonella sakaiensis or Comamonas sp. strain E6. Engineering strategies included codon optimization, genomic integration and epigenomic expression through plasmids with bicistronic expression units. Despite the successful expression, no functional conversion of TPA to PCA was detected. We hypothesize that one of the primary roadblocks may be the maturation of TPA-DO, a Rieske non-heme iron dioxygenase that requires a specific iron-sulfur (Fe-S) cluster for functioning. Despite genetic interventions to disrupt iron homeostasis, the enzymatic pathway remained inactive. These findings highlight a fundamental incompatibility between bacterial Fe-S enzyme requirements and the yeast cytosolic protein assembly machinery, suggesting that TPA upcycling in yeast demands further fundamental breakthroughs.