High-Throughput Promoter Engineering of Vacuolar-Associated Genes Enhances Extracellular Betanin Accumulation in Yarrowia lipolytica.

Betanin is a natural red pigment that accumulates within plant vacuoles. It is hypothesized that vacuolar sequestration could also restrict the extracellular accumulation of heterologously produced betanin in the yeast Yarrowia lipolytica. To investigate the role of vacuolar functions in influencing extracellular betanin levels, we employed the TUNEYALI platform to generate a promoter-engineering library targeting 21 vacuole-associated genes. Screening 470 transformants identified nine variants with enhanced extracellular betanin accumulation. Whole-genome sequencing revealed that all nine isolates harbored disruptions in YALI1_C31684g, the gene most closely related to Saccharomyces cerevisiaeVPS8, which encodes a CORVET-specific tethering subunit. Reverse engineering confirmed that partial knockdown of YALI1_C31684g led to a 1.18-fold increase in total betanin production and a 61.7% increase in extracellular betanin levels, whereas complete deletion of the gene reduced extracellular accumulation. Quantitative PCR analysis showed that the promoter knockout construct maintained approximately 47% of wild-type transcript levels, suggesting that a partial loss of function is more effective than a complete loss of function in enhancing extracellular betanin. Confocal microscopy demonstrated extensive vacuolar fragmentation in the knockdown strain, with the highest levels of extracellular betanin correlating with the most pronounced fragmentation phenotype. Collectively, these findings identify YALI1_C31684g as a key regulator of extracellular betanin accumulation in Y. lipolytica and demonstrate that modulating vacuolar trafficking functions can enhance the extracellular fraction of metabolites stored in vacuoles.