NRPS-like Gene <i>LYS2</i> Contributed to the Biosynthesis of Cyclo(Pro-Val) in a Multistress-Tolerant Aromatic Probiotic, <i>Meyerozyma guilliermondii</i> GXDK6

Cyclo(Pro-Val) is a diketopiperazine (DKP) found widespread in marine microbes and resulting food products. With new bioactivities of cyclo(Pro-Val) being continually discovered, its potential applications in agriculture and food are becoming more evident, highlighting the need for efficient and practical methods to produce these compounds. However, the biosynthesis mechanisms of cyclo(Pro-Val), particularly in probiotics, remain unclear, and the functional identification of nonribosomal peptide synthases (NRPS) is still limited. This study presents a new efficient cyclo(Pro-Val) production system using the marine probiotic <i>Meyerozyma guilliermondii</i> (<i>M. guilliermondii</i>) GXDK6 as a chassis cell. We further characterized an NRPS-like gene, <i>LYS2</i>, which spans 3825 bp and encodes an l-2-amino-hexanedioic acid reductase (Lys2p) consisting of 1274 amino acids. A novel macrocyclization mechanism was revealed involving the peptide N-terminal and C-terminal imines mediated by Lys2p through nucleophilic reactions. Overexpressing <i>LYS2</i> in GXDK6 resulted in a 45.5% increase in cyclo(Pro-Val) production, while knockout <i>LYS2</i> completely halted its synthesis. This study revealed a novel metabolic regulatory pathway and biosynthetic mechanism for cyclo(Pro-Val) in marine yeast <i>M. guilliermondii</i> GXDK6. Refactoring the biosynthetic pathway in yeast through <i>LYS2</i> provides an alternative approach for producing cyclo(Pro-Val).