Unveiling a novel uronate dehydrogenase from industrial wastewater metagenomes for efficient galactaric acid production in engineered Saccharomyces cerevisiae

Abstract

Galactaric acid is a valuable chemical utilized in the cosmetic and polymer industries. This platform chemical can be produced through a single enzymatic step catalyzed by uronate dehydrogenase (UDH) from D-galacturonic acid, which can be derived from the hydrolysis of pectin-rich feedstock. However, most characterized UDHs have been sourced from cultured microorganisms, leaving a significant gap in our understanding of UDHs from uncultured microbial communities. In this study, we identified and characterized three novel UDHs from the metagenomes of industrial wastewater. Following activity screening, the m2UDH was selected due to its superior enzyme activity in a Saccharomyces cerevisiae host. Through semi-rational protein engineering, the m2UDH^T123V variant was generated with 13-fold increase in enzyme activity compared to the wild-type enzyme. Engineered thermotolerant S. cerevisiae expressing m2UDH^T123V variants exhibited approximately 2.8-fold higher enzymatic activity and 2.4-fold higher galactaric acid production at 40 °C compared to the wild-type enzyme. Thus, this study highlights the significance of exploring diverse metagenomic environments for enzyme discovery and presents a promising approach for the bioprocessing of pectin-rich feedstocks into value-added biochemicals.