Transcriptomics‐Driven Identification and Engineering of a Novel Lactone Esterase ( Sr LE) for Efficient Sophorolipid Lactonization in the Yeast Starmerella riodocensis

ABSTRACT Lactonic sophorolipids (LSLs) are high‐value glycolipid biosurfactants with superior bioactivities, yet their industrial application is hampered by the low lactonization efficiency of wild‐type strains. In this study, we employed a transcriptomics‐driven approach to decode the regulatory landscape of the underexplored yeast Starmerella riodocensis GT‐SL1R and engineer it for high‐titer LSL production. Comparative transcriptome analysis under nitrogen limitation revealed a massive metabolic reprogramming, identifying 497 upregulated genes, including the core SL biosynthetic cluster ( CYP52M1 , ugta1 , ugtb1 , at , and mdr1 ). Crucially, we identified a novel lactone esterase ( Sr LE) with only 49% similarity to the well‐known Sb LE from Starmrella bombicola . While growth‐related pathways (ribosome biogenesis and DNA replication) were repressed, nitrogen recycling mechanisms were activated to fuel SL biosynthesis. Directed metabolic engineering of the lactonization pathway through SrLE or SbLE overexpression, coupled with fed‐batch fermentation in a 5‐L bioreactor, achieved exceptional LSL titers of 189.33 g·L −1 and 191.45 g·L −1 , respectively. Conversely, SrLE knockout resulted in the exclusive production of acidic SLs, confirming its pivotal role in lactonization. These results provide deep mechanistic insights into SL biosynthesis and demonstrate the power of targeted enzyme engineering in creating robust microbial cell factories for specialized glycolipid production.