Systematic Engineering for Efficient Uric Acid-Degrading Activity in Probiotic Yeast Saccharomyces boulardii

Hyperuricemia, caused
by uric acid disequilibrium, is a prevalent
metabolic disease that most commonly manifests as gout and is closely
associated with a spectrum of other comorbidities such as renal disorders
and cardiovascular diseases. While natural and engineered probiotics
that promote catabolism of uric acid in the intestine have shown promise
in relieving hyperuricemia, limitations in strain efficiency and the
requirements for achieving high performance remain major hurdles in
the practical application of probiotic-mediated prevention and management.
Here, we employed a systematic strategy to engineer a high-efficiency
uric acid catabolism pathway in S. cerevisiae. An uricase from Vibrio vulnificus, exhibiting high-level activity in S. cerevisiae, was identified as the uric acid-degrading component. The expression
level and stability of urate transporter UapA were improved by constructing
a chimera, enabling reliable uric acid import in S.
cerevisiae. Additionally, constitutive promoters were
selected and combinatorially assembled with the two functional components,
creating a collection of pathways that confer varied levels of uric
acid catabolic activity to S. cerevisiae. The best-performing pathway can express uric acid-degrading activity
up to 365.32 ± 20.54 μmol/h/OD, requiring only simple cultivation
steps. Eventually, we took advantage of the genetic similarity between
model organism S. cerevisiae and probiotic S. boulardii and integrated the optimized pathway
into identified high-expression integration loci in the S. boulardii genome. The activity can be stably maintained
under high-density fermentation conditions. Overall, this study provided
a high-potential hyperuricemia-managing yeast probiotic strain, demonstrating
the capabilities of developing recombinant probiotics.