[Cloning of cytochrome P450 reductase genes from Carthamus tinctorius and comparison of electron donation activity].

Cytochrome P450(CYP450) reductase(CPR), as the electron donor of CYP450, is a key component supporting CYP450-catalyzed structural modification of bioactive plant compounds such as flavonoids. In Carthamus tinctorius, CPR is integral to the biosynthesis of bioactive flavonoid compounds, including hydroxysafflor yellow A. Based on the analysis of transcriptomic data from different parts of C. tinctorius, this study cloned three CPR genes-CtCPR1, CtCPR2, and CtCPR3-with open reading frames of 2 133, 2 133, and 2 124 base pairs, respectively, which encoded proteins of 710, 710, and 707 amino acids. Bioinformatic characterization predicted molecular masses of 78.9, 79.0, and 78.4 kDa, theoretical isoelectric points of 5.26, 5.32, and 5.35, and negative grand average of hydropathicity scores, consistent with a hydrophilic nature. Sequence alignment and phylogenetic analysis revealed conserved domains of flavin adenine dinucleotide, flavin mononucleotide, and nicotinamide adenine dinucleotide phosphate in CtCPR1, CtCPR2, and CtCPR3, which clustered with AtCPR2 of Arabidopsis thaliana in the Class Ⅱ CPR subfamily. The three CPR genes were individually combined with the functionally known ent-kaurene oxidase AtKO gene and C. tinctorius derived CtKO gene and then transformed into an ent-kaurene-producing yeast strain for fermentation. The electron donation activities were compared based on the yield of the fermentation product, ent-kaurenoic acid. The results showed that CtCPR1, CtCPR2, and CtCPR3 all had electron donation activity. When co-expressed with CtKO, CtCPR1 exhibited enhanced electron donation activity, whereas CtCPR3 demonstrated superior electron donation activity when paired with AtKO, which highlighted the high partner compatibility between CPR and CYP450. This study not only provides novel genetic resources for the efficient production of bioactive C. tinctorius compounds through synthetic biology approaches, but also contributes to elucidating the metabolic regulatory networks of flavonoids in plants.