The key enzymes involved in the flavonoid bio-synthesis pathway have been extensively studied in seed plants, but relatively less in ferns. In this study, two 4-Coumarate: coenzyme A ligases (Sc4CL1 and Sc4CL2) and one novel chalcone synthase (ScCHS1) were functionally charac-terized by mining the Stenoloma chusanum tran-scriptome database. Recombinant Sc4CLs were able to esterify various hydroxycinnamic acids to corresponding acyl-coenzyme A (CoA). ScCHS1 could catalyze p-coumaroyl-CoA, cinnamoyl-CoA, caffeoyl-CoA, and feruloyl-CoA to form naringenin, pinocembrin, eriodictyol, and homoeriodictyol, respectively. Moreover, enzymatic kinetics studies revealed that the optimal substrates of ScCHS1 were feruloyl-CoA and caffeoyl-CoA, rather than p-coumaroyl-CoA, which was substantially different from the common CHSs. Crystallographic and site-directed mutagenesis experiments indicated that the amino acid residues, Leu87, Leu97, Met165, and Ile200, located in the substrate-binding pocket near the B-ring of products, could exert a significant im-pact on the unique catalytic activity of ScCHS1. Furthermore, overexpression of ScCHS1 in tt4 mu-tants could partially rescue the mutant phenotypes. Finally, ScCHS1 and Sc4CL1 were used to synthe-size flavanones and flavones with multi-substituted hydroxyl and methoxyl B-ring in Escherichia coli, which can effectively eliminate the need for the cy-tochrome P450 hydroxylation/O-methyltransferase from simple phenylpropanoid acids. In summary, the identification of these important Stenoloma enzymes provides a springboard for the future production of various flavonoids in E. coli.

Key Laboratory of Chemical Biology of Natural Products,Ministry of Education,School of Pharmaceutical Sciences,Shandong University,Jinan 250100,China;National Key Laboratory of Plant Molecular Genetics,Center for Excellence in Molecular Plant Sciences,Shanghai Institute of Plant Physiology and Ecology,Chinese Academy of Sciences,Shanghai 200032,China

[1]Rong Ni;Meng Niu;Jie Fu;Hui Tan;Ting-Ting Zhu;Jing Zhang;Hong-Xiang Lou;Peng Zhang;Jian-Xu Li;Ai-Xia Cheng-.Molecular and structural characterization of a promiscuous chalcone synthase from the fern species Stenoloma chusanum)[J].植物学报（英文版）,2022(10):1935-1951

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