Background/Objectives: Ulp1 is a vital regulator of the cell cycle, with its absence leading to G2/M phase arrest in Saccharomyces cerevisiae. This study aims to investigate the role of Ulp1 in cell cycle regulation in Pichia pastoris and to elucidate its mechanisms of action, particularly through the modulation of the gene INO1. Methods: We generated Ulp1 knockout strains in Pichia pastoris using the FLP-FRT system and performed RNA sequencing (RNA-seq) to analyze gene expression changes. We assessed cell proliferation in Ulp1 knockout and INO1 overexpressing strains, as well as the effects of inositol supplementation. Results: Our findings revealed significant downregulation of INO1 and other genes in Ulp1 knockout strains. Importantly, overexpression of INO1 restored cell proliferation, indicating that Ulp1 regulates this process via INO1. Notably, supplementation with exogenous inositol did not rescue cell proliferation, suggesting that the enzymatic activity of INO1 is not required for Ulp1’s regulatory function. Conclusions: This study demonstrates that Ulp1 modulates cell proliferation in Pichia pastoris through INO1, independent of its enzymatic activity. These insights enhance our understanding of Ulp1’s role in cell cycle regulation and open new avenues for exploring the molecular mechanisms governing yeast cell division. Further investigations are warranted to delineate the intricate regulatory pathways involved in this process.

Pichia pastoris is extensively employed as a host for expressing foreign proteins, and its secretion system is especially advantageous for efficient protein purification. Ulp1 is essential for the cell cycle. In Saccharomyces cerevisiae, the absence of Ulp1 leads to G2/M cell cycle arrest. Similarly, in Pichia pastoris, we also observed G2 phase stalling in Ulp1 knockout cells. RNA-seq data revealed significant changes in seven genes, including INO1. Interestingly, overexpressing INO1 in Ulp1 knockout cells restored cell proliferation, suggesting that Ulp1 regulates cell proliferation through INO1. However, adding extra inositol to Ulp1 knockout cells was not able to restore cell prolifera-tion, indicating that the enzyme activity of INO1 is not essential for cell proliferation. Our data provide new insights into Ulp1 functions in the cell cycle and raise new questions about yeast cell cycle regulation.