Genome-Wide Identification and Functional Analysis of the Norcoclaurine Synthase Gene Family in Aristolochia contorta

<i>Aristolochia contorta</i> Bunge has been widely used as traditional Chinese medicine materials. However, its utility faces a great challenge due to the presence of aristolochic acids (AAs), a class of benzylisoquinoline alkaloid (BIA) derivatives. The first step in BIA skeleton formation is catalysis by norcoclaurine synthase (NCS). To gain knowledge of BIA and AA biosynthesis in <i>A. contorta</i>, genome-wide characterizations of <i>NCS</i> genes were carried out. This resulted in the identification of 15 <i>A. contorta NCSs</i>, namely, <i>AcNCS1-AcNCS15</i>. The AcNCS1-AcNCS8 proteins contained one catalytic domain, whereas the AcNCS9-AcNCS15 proteins had two. Phylogenetic analysis shows that AcNCS proteins can be classified into two clades. Gene expression analysis shows that five <i>AcNCSs</i>, including <i>AcNCS2</i>, <i>AcNCS4</i>, <i>AcNCS5</i>, <i>AcNCS14</i>, and <i>AcNCS15</i>, exhibited relatively high expression in roots and flowers, where norcoclaurine accumulated. An enzyme catalytic activity assay shows that all five of the AcNCSs can catalyze norcoclaurine formation with AcNCS14 and AcNCS15, exhibiting higher catalytic efficiency. Precolumn derivatization analysis shows that the formed norcoclaurine included (<i>S</i>)- and (<i>R</i>)-norcoclaurine, with more (<i>S</i>)-configuration. The results provide useful information for further understanding BIA and AA biosynthesis in <i>A. contorta</i> and for AA elimination and bioactive compound improvement in AA-containing medicinal materials.