A systematic study of regulating inorganic polyphosphates production in Saccharomyces cerevisiae

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, plays critical roles in diverse biological processes spanning blood coagulation, immunomodulation, and post-translational protein modifications in eukaryotes. Notably, long-chain polyP (>100 phosphate units) exhibits distinct biological functionalities compared to shorter-chain counterparts. While Saccharomyces cerevisiae serves as a promising microbial platform for polyP biosynthesis, the genetic regulatory mechanisms underlying polyP metabolism remain poorly elucidated. Here, we systematically investigated the genetic determinants governing intracellular polyP levels and chain length dynamics in yeast. Through screening a library of 55 single-gene knockout strains, we identified six mutants (Δddp1, Δvip1, Δppn1, Δppn2, Δecm33, and Δccr4) exhibiting elevated polyP accumulation, whereas deletions of vtc1, kcs1, vma22, vma5, pho85, vtc4, vma2, vma3, ecm14, and vph2 resulted in near-complete polyP depletion. Subsequent combinatorial deletions in the Δppn1 background revealed that the Δppn1Δvip1 double mutant achieved synergistic enhancement in both polyP concentration (53.01 mg-P/g-DCW) and chain length, attributable to increased ATP availability and reduced polyphosphatase activity. Leveraging CRISPR/Cas9-mediated overexpression in Δppn1Δvip1, we engineered strain PP2 (vtc4 overexpression), which demonstrated a 2-fold increase in polyP yield (62.6 mg-P/g-DCW) relative to wild-type BY4741, with predominant synthesis of long-chain species. Mechanistically, qRT-PCR analysis confirmed that PP2 exhibited 46-fold up-regulation of vtc4 coupled with down-regulation of polyphosphatases encoding genes, ppn2, ddp1, and ppx1. This study performed a systematic study of regulating inorganic polyphosphates production in yeast and provides a synthetic biology strategy to engineer high-yield polyP-producing strains, advancing both fundamental understanding and biotechnological applications.