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

Abstract

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.

Keywords: Saccharomyces boulardii; engineered probiotic; hyperuricemia; urate transporter; uric acid degradation; uricase.

1

Design of probiotic yeast for uric acid-degradation. (A) General diagram of engineered uric acid-degradation in probiotic yeast S. boulardii. (B) Identification of the urate oxidase exhibiting high-level activity in Saccharomyces chassis. Expression cassettes of candidate UOX CDS regulated by the TDH3 promoter and the PGK1 terminator were cloned into a CEN expression plasmid and transformed into S. cerevisiae. The UOX-expressing recombinant strains were cultured for 16 h and lysed, and the supernatant was collected. The crude enzyme activities and total protein concentrations of the supernatant were determined, and specific activities were calculated. The phylogenetic tree was generated based on protein sequences of the tested UOXs. BY4741, lysate supernatant of S. cerevisiae BY4741 strain; BY4741+pPOT, lysate supernatant of BY4741 harboring empty vehicle plasmid; pPOT+0.1U, lysate supernatant of BY4741 harboring empty vehicle plasmid with 0.1U commercial uricase. All tested UOXs are listed in Table S3. Data presented was obtained from biological triplicates.

2

Establishment and improvement of uric acid uptake in Saccharomyces chassis. (A) Coexpression of UOX_Vvul and UapA confer whole cell UAD ability to S. cerevisiae. Expression cassettes of UOX_Vvul regulated by the TDH3 promoter and the PGK1 terminator and UapA regulated by the EFB1 promoter and the ADH1 terminator were cloned into the same CEN expression plasmid and transformed into S. cerevisiae. BY4741, S. cerevisiae BY4741 strain; pPOT, BY4741 harboring empty vehicle plasmid; pPOT-UOX, BY4741 harboring plasmid carrying UOX_Vvul expression cassette; pPOT-UapA, BY4741 harboring plasmid carrying UapA expression cassette; pCCU-UOX+UapA, BY4741 harboring plasmid carrying UOX_Vvul and UapA coexpression cassettes. The recombinant strain was cultured for 16 h, and 0.5 OD of cells was used for the UAD assay at 30 °C. (B) The whole cell UAD activity decreases at 37 °C compared to at 30 °C. The recombinant strain was cultured at 30 °C for 16 h, and 0.5 OD of cells was used for the UAD assay at 30 and 37 °C incubation temperatures. (C) The UapA_GPA1Nt chimera increases the UapA level at 30 and 37 °C. Expression cassettes of UapA WT and chimera (Table S5) fused with GFP protein under the regulation of the PGK1 promoter and the ADH1 terminator were cloned into a CEN expression plasmid and transformed into S. cerevisiae. The recombinant strains were cultured at 30 °C for 14 h, and the cultures were divided and incubated for 2 h at 30 and 37 °C, before GFP intensities were quantified and normalized to OD₆₀₀ readings. (D) Coexpression of UapA_GPA1Nt chimera with UOX_Vvul improves whole cell UAD activity at 37 °C. The recombinant strains coexpressing UOX_Vvul with UapA WT or UapA variants were cultured at 30 °C for 16 h, and 0.5 OD of cells was used for the UAD assay at 30 and 37 °C incubation temperature. Crude enzyme assays in this figure were performed as described above. *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001. Data presented was obtained from biological triplicates.

3

Combinatorial optimization of urate oxidase and urate transporter activity improves pathway activity. UOX_Vvul and UapA_GPA1Nt encoding sequences were assembled with five strong constitutive promoters (pTDH3, pPDC1, pPGK1, pTEF2, and pCCW12) and three terminators (tSCW4, tPGK1, and tADH1) in a combinatorial manner utilizing a hierarchical assembly system modified from the YeastFab method. The receiver plasmids carrying the resulting pathways were transformed into S. cerevisiae. Verified clones were cultured for 16 h, and 0.5 OD of cells was used for whole cell UAD activity assay at 37 °C. The data presented here were derived from pathways that do not contain repeating regulatory elements. A complete set of the data can be found in Table S6. Data presented were based on biological triplicates.

4

Identification of genome integration sites that support high-level UAD activity expression in probiotic yeast chassis. (A) Evaluation of high-expression genomic integration sites in S. boulardii. An optimized pathway was integrated into selected genomic sites through homologous recombination and uracil auxotroph selection (Table S7). Whole cell UAD activities were determined and normalized. (B) Efficient degradation of uric acid by integrating of optimized pathway in S. boulardii. BY4741+P77, S. cerevisiae BY4741 strain with a plasmid carrying pathway#77; MYA796+P77, S. boulardii MYA796 strain with a plasmid carrying pathway#77; MYA796+Int 11, S. boulardii MYA796 strains expressing the P77 pathway integrated at testing genomic integration loci 11. Strains were cultured for 16 h, and 0.5 OD of cells was used for whole cell UAD activity assay at 37 °C. *, P < 0.05. Data presented were based on biological triplicates.