Screening and Engineering of Hyperactive Yeast Aquaporins for Xylitol Transport Using Biosensors

Microbial cell factories provide a sustainable platform for producing valuable compounds from renewable substrates. However, the transport of metabolites-particularly the import of substrates and the export of products-remains a major bottleneck in improving production efficiency. Aquaglyceroporins (AQGPs), a subgroup of aquaporins, are small, structurally conserved and energy-independent membrane channels that represent promising targets to overcome this limitation. Yeast AQGPs, known as Fps1, contain extended N- and C-terminal cytosolic regions that play essential roles in channel regulation. Previous studies have suggested that deletion of short conserved motifs within these termini can render Fps1 hyperactive, but this has only been studied in yeast systems and primarily in the context of glycerol. We further explored this phenomenon in the context of xylitol transport, and developed an E. coli-based biosensor to evaluate AQGP-mediated xylitol import. Using this biosensor, truncated variants of Fps1 homologs from Saccharomyces cerevisiae, Kluyveromyces marxianus and Yarrowia lipolytica were evaluated. Two hyperactive variants, ScFps1.4 and KmFps1.2, were identified, revealing aquaglyceroporin- and host-dependent effects on AQGP activity. These findings provide new insights into aquaglyceroporin regulation and its potential for improving metabolite transport in microbial cell factories.