Functional characterization of loganic acid O-methyltransferase and secologanin synthase from Uncaria rhynchophylla and two-step biosynthesis of secologanin in tobacco

Rhynchophylline (RIN) and isorhynchophylline (IRN) have been evaluated as the pivotal medicinal ingredients in Uncaria rhynchophylla. They display excellent antihypertensive and neuroprotective effects. But in U. rhynchophylla , the gene elements responsible for the biosynthesis of secologanin, a critical precursor for RIN and IRN, remain unidentified. In this study, one UrLAMT gene and three UrCYP72A genes ( UrCYP72A1177 , UrCYP72A1178 , and UrCYP72A1179 ) are mined and cloned from U. rhynchophylla . Biochemical verification reveals that UrLAMT exhibits C11-carboxy O -methyltransferase activity for loganic acid and UrCYP72A1177–UrCYP72A1179 display C7-C8 bond scission activity for loganin. Evolutionary origin analyses imply that the genes responsible for iridoid biosynthesis cluster in adjacent region in O. pumila and L. japonica , but UrLAMT and UrCYP72A1178 – UrCYP72A1179 distribute scatteredly in U. rhynchophylla . Kinetic parameter analyses reveal that UrCYP72A-catalyzed C7-C8 scission is a rate-limiting step. The enzymatic activities of UrCYP72A1177–UrCYP72A1179 are significantly improved by D396E, F327G + P393V, and L321G + V390I mutation, respectively. Two-step biosynthesis of secologanin is facilitated by co-expression of UrLAMT and UrCYP72As in tobacco. The highest secologanin yield (3.35 μg g −1 ) in tobacco is achieved in UrLAMT and UrCYP72A1177-D396E co-expression group. This study newly uncovers two adjacent biosynthetic steps for secologanin in a RIN-producing plant, proposes a plausible mechanism for UrCYP72A-mediated C C bond cleavage, and boosts the secologanin yield in tobacco. These findings would accelerate the schedule of RIN biomanufacture.