Engineering xanthophyll esterification: LbXES2 mediates zeaxanthin dipalmitate biosynthesis and remodels fruit flavor metabolism

Abstract Lycium barbarum fruits are valued for bioactive carotenoids, yet the enzymatic basis underlying accumulation of their hallmark xanthophyll diester, zeaxanthin dipalmitate (ZD), remains unclear. Biosynthesis of ZD requires two sequential reactions: β-carotene hydroxylation catalyzed by β-carotene hydroxylases (BCHs) to produce zeaxanthin, followed by xanthophyll esterification mediated by xanthophyll ester acyltransferases (XESs). However, the specific genes responsible for ZD formation in L. barbarum have not been functionally defined. Here, we integrated genome mining, phylogenetics, developmental metabolite profiling, tissue- and stage-resolved expression analysis and functional validation using Escherichia coli complementation, transient expression in L. barbarum and tobacco, and fruit-specific co-expression in tomato. Carotenoid profiling revealed a ripening-associated transition from free xanthophylls to esters. Among the candidates, LbBCH1 and LbXES2 showed fruit-preferential expression, positively correlated with ZD accumulation, and both encoded plastid-localized proteins. In E. coli complementation assays, LbBCH1 converted β-carotene to zeaxanthin, and LbXES2 further catalyzed the formation of ZD from zeaxanthin. Transient expression assays showed that LbXES2 could esterified multiple xanthophylls and exhibited a preference for palmitoylation preference, and ZD production was enhanced by LbBCH1-mediated zeaxanthin supply. In tomato, fruit-specific co-expression of LbBCH1 and LbXES2 enabled de novo xanthophyll ester biosynthesis, elevated total carotenoids, promoted chromoplast/plastoglobule development, and altered sugars, organic acids and carotenoid-derived volatiles. Collectively, these results identify LbXES2 as a xanthophyll esterification enzyme associated with ZD biosynthesis and support a functional role for LbBCH1-mediated zeaxanthin supply in this process. Our study provides molecular tools and a heterologous engineering strategy for producing high-value esterified carotenoids in fleshy fruits.