Biosynthesis of cannflavins A and B from Cannabis sativa L Author Rea, Kevin A ∗ & Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N 1 G 2 W 1, Canada Author Casaretto, José A. Author Al-Abdul-Wahid, M. Sameer Author Sukumaran, Arjun Author Geddes-McAlister, Jennifer Author Rothstein, Steven J. Author Akhtar, Tariq A. text Phytochemistry 2019 2019-05-28 164 162 171 http://dx.doi.org/10.6084/m9.figshare.24741938.v1 journal article 284782 10.1016/j.phytochem.2019.05.009 b6b9be71-bc68-4ec7-a82e-93da86a5eccd 1873-3700 10483096 2.1. Phylogenetic analysis of C. sativa prenyltransferases To synthesize cannflavins A and B, a prenyl moiety must be added to position 6 of a flavone that typically accumulates in C. sativa . Therefore, we first searched for gene sequences that were putatively annotated as flavonoid or related aromatic prenyltransferases in the Transcriptome Shotgun Assembly (TSA) database for C. sativa , which is accessible through NCBI. A previously described flavone prenyltransferase from Glycyrrhiza uralensis (GuA6DT; GenBank AIT11912.1) was used as a query in these searches ( Li et al., 2014 ). GuA6DT prenylates apigenin which is a widespread plant flavone that also accumulates in C. sativa ( McPartland and Russo, 2001 ) . This search uncovered eight full-length cDNA sequences from C. sativa that exhibited 22–53% identity at the amino acid level to GuA6DT and were putatively annotated as C. sativa prenyltransferases ( CsPT1-8 ; Fig. S1 ). One of the prenyltransferases that were identified in this search ( CsPT1 ) matched a previously reported enzyme from C. sativa that is known to be involved in the prenylation of olivetolic acid to cannabigerolic acid in the cannabinoid biosynthesis pathway ( Page and Boubakir, 2014 ). We next performed a phylogenetic analysis that included CsPT1 and these seven other prenyltransferases from C. sativa along with all known plant prenyltransferases that have been previously shown to accommodate aromatic substrates ( Fig. 1 ; Fig. S1 ). This analysis demonstrated that plant aromatic prenyltransferases fall into six distinct groups, which are conveniently defined by the specific branch of aromatic metabolism in which they participate. The eight CsPTs occupy three of these six groups: CsPT2 and CsPT6 reside in a unique clade of prenyltransferases (Group 2) which have been shown to participate in the tocopherol biosynthetic pathway ( Collakova and DellaPenna, 2001 ; Savidge et al., 2002 ; Tian et al., 2007 ). CsPT5 appears to be orthologous to homogentisate solanesyltransferases (Group V) that function in plastoquinone biosynthesis ( Venkatesh et al., 2006 ; Tian et al., 2007 ). The five remaining CsPTs ( CsPT1, 3 , 4 , 7 , and 8 ) formed a third and distantly related group (Group VI) that includes two prenyltransferases from Humulus lupulus (hops), which are involved in the aromatic prenylation reactions required for terpenophenolic biosynthesis ( Nagel et al., 2008 ; Tsurumaru et al., 2012 ; Li et al., 2015 ). Surprisingly, this analysis revealed that none of the CsPTs were closely related to any of the flavonoid or coumarin prenyltransferases (Groups I and IV, respectively) that have been previously identified in various plant species ( Sasaki et al., 2008 , 2011 ; Akashi et al., 2009 ; Shen et al., 2012 ; Wang et al., 2014 ; Munakata et al., 2016 ; Yoneyama et al., 2016 ; Yang et al., 2018 ). Interestingly, in silico analysis of each CsPT predicted that they are all targeted to plastids ( Table S2 ). We return to this point below.