Co-expression of GTP cyclohydrolase I and aminodeoxychorismate synthase from alfalfa improves folate accumulation and plant growth in Lotus corniculatus

Folate (vitamin B9) is an essential nutrient for livestock. However, natural folate levels in forage crops are often insufficient to meet livestock nutritional requirements. Metabolic engineering offers a promising solution by enhancing folate biosynthesis in plants. In this study, we engineered Lotus corniculatus , an important legume forage, to enhance folate biosynthesis by co-expressing GTP cyclohydrolase I and aminodeoxychorismate synthase genes MsGCHI and MsADCS from alfalfa ( Medicago sativa ). In T 0 transgenic plants grown under greenhouse conditions, total folate levels reached 2.1-fold higher in leaves and 1.7-fold higher in stems compared to WT, with similar trends in field trials, corresponding increases of 1.2-fold and 1.6-fold were observed. The elevated folate was accompanied by increased pools of biosynthetic precursors (pteridines and free p-ABA) and multiple folate derivatives, including 5-methyltetrahydrofolate (5-M-THF), 5,10-Methylenetetrahydrofolate (5,10-CH=THF), 5-Formyltetrahydrofolate (5-F-THF), tetrahydrofolate (THF)-related pool, and dihydrofolate (DHF). Furthermore, RT-qPCR analysis revealed that the co-expression of MsGCHI and MsADCS up-regulated the expression of key genes ( ADCL , HPPK/DHPS , DHFR , DHFS , DHNA , FPGS ) involved in folate biosynthesis pathway of transgenic L. corniculatus , indicating a coordinated metabolic response to increased precursor availability. In addition to biochemical modifications, phenotypic characterization of transgenic plants revealed enhanced vegetative growth, including increased plant height, leaf area, branches, and biomass under greenhouse and/or field conditions. This study demonstrates the feasibility of folate biofortification in a forage legume and provides a proof of concept for linking enhanced folate metabolism with improved crop productivity in an agronomic context. These findings offer a foundational reference for future efforts toward developing biofortified forage as a potential nutritional supplementation strategy. • Co-expressing MsGCHI and MsADCS elevates folates in L. corniculatus via enhanced precursors and gene upregulation. • Transgenic plants exhibit improved height, biomass, and root growth in greenhouse/field conditions. • Study establishes high-yield, folate-biofortified forage with optimized metabolic and agronomic traits.