In Vivo Functional Investigation of LysC3 (Aspartokinase 3) Revealed Its Role in Priming the Biosynthesis of 1-Deoxynojirimycin in Mulberry

Aspartokinase (LysC) has been proposed as a key enzyme involved in 1-deoxynojirimycin (1-DNJ) biosynthesis in mulberry, yet direct functional evidence remains limited. In this study, we cloned and functionally characterized MmLysC3, a candidate aspartokinase gene from mulberry (Morus multicaulis), and investigated its role in 1-DNJ biosynthesis. Domain architecture analysis confirmed the presence of both the conserved AA_kinase catalytic domain and two ACT regulatory domains, placing MmLysC3 within the class I aspartokinase family. Expression profiling revealed that MmLysC3 exhibits tissue-preferential expression, with higher transcript levels in buds and young leaves, spatially coinciding with active 1-DNJ accumulation sites. Correlation analysis across ten mulberry varieties demonstrated a significant positive association between LysC3 expression and 1-DNJ content (Spearman’s r = 0.6743, p = 9.04 × 10−5). Critically, transient overexpression of MmLysC3 in mulberry leaves resulted in a marked increase in 1-DNJ content, with the highest-accumulating line reaching approximately twice that of the control. These findings provide the first functional evidence that LysC3 acts as a positive rate-limiting enzyme in the aspartate-derived branch of the 1-DNJ biosynthetic pathway in mulberry, bridging the gap between transcriptomic predictions and functional validation.