title: Biosynthesis of homoeriodictyol from eriodictyol by flavone 3'-O-methyltransferase from recombinant Yarrowia lioplytica: Heterologous expression, biochemical characterization, and optimal transformation.
abstract: In this work, we attempted to synthesize homoeriodictyol by transferring one methyl group of S-adenosyl-L-methionine (SAM) to eriodictyol using flavone 3'-O-methyltransferase ROMT-9, which was produced by recombinant Yarrowia lipolytica. Specifically, the ROMT-9 gene from rice was synthesized and cloned into the multi-copy integrative vector pINA1297, and was further expressed in Y. lipolytica with a growth phase-dependent constitutive promoter hp4d. The highest ROMT-9 activity reached 5.53 U/L after 4 days of culture in shake flask. The optimal pH and temperature of the purified ROMT-9 were 8.0 and 37 °C, respectively. The purified enzyme was stable up to 40 °C, and retained more than 80% of its maximal activity between pH 6.5 and 9.0. The recombinant ROMT-9 did not require Mg²⁺ for catalysis, while was completely inhibited in the presence of 5 mM Zn²⁺, Cu²⁺, Ba²⁺, Al³⁺, or Ni²⁺. The purified ROMT-9 was used to synthesize homoeriodictyol, and the maximal transformation ratio reached 52.4% at 16 h under the following conditions: eriodictyol 0.2 g/L, ROMT-9 0.16 g/L, SAM 0.2 g/L, CH₃OH 6% (v/v), temperature 37 °C, and pH 8.0. This work provides an alternative strategy for efficient synthesis of homoeriodictyol and compared to the traditional plant extraction or chemical synthesis, the biotransformation approach generates less environmental pollution and has a great potential for the sustainable production of homoeriodictyol.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Qingtao
authors.initials: Q
authors.lastname: Liu
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Jingwen
authors.initials: J
authors.lastname: Zhou
authors.forename: Hyun-Dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Chen
authors.forename: Catherine
authors.initials: C
authors.lastname: Madzak
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D044950:Flavanones / Q000378:metabolism*; D047309:Flavones / Q000096:biosynthesis*; D015870:Gene Expression; D005822:Genetic Vectors; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D044950:Flavanones; D047309:Flavones; D011994:Recombinant Proteins; D008780:Methyltransferases; C503231:homoeriodictyol; C007619:eriodictyol
keywords: <rk-italic>Yarrowia lipolytica</rk-italic>; Biotransformation; Flavone 3′-O-methyltransferase; Heterologous expression; Homoeriodictyol