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.

中文翻译：

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重组解脂耶氏酵母中的黄酮3'-O-甲基转移酶从香茅酚生物合成高香茅酚：异源表达，生化特性和最佳转化。

在这项工作中，我们尝试使用黄酮3'-O-甲基转移酶ROMT-9（由解脂耶氏酵母（Yarrowia lipolytica）生产）将S-腺苷-L-甲硫氨酸（SAM）的一个甲基转移至芥酸中来合成高芥酸。具体而言，合成了来自水稻的ROMT-9基因，并将其克隆到多拷贝整合型载体pINA1297中，并在脂解耶氏酵母中进一步表达，且具有生长阶段依赖性组成型启动子hp4d。在摇瓶中培养4天后，最高的ROMT-9活性达到5.53 U / L。纯化的ROMT-9的最佳pH和温度分别为8.0和37°C。纯化的酶在高达40°C的温度下是稳定的，并且在pH 6.5和9.0之间保留了其最大活性的80％以上。重组ROMT-9不需要Mg²⁺进行催化，而在5 mM Zn 2+，Cu 2+，Ba 2+，Al 3+或Ni 2+的存在下则被完全抑制。纯化后的ROMT-9用于合成高顺丁烯二酚，在以下条件下在16 h时最大转化率达到52.4％：顺丁烯二酚0.2 g / L，ROMT-9 0.16 g / L，SAM 0.2 g / L，CH 3 OH 6％ （v / v），温度37°C和pH 8.0。这项工作提供了一种高效合成高茴香油的替代策略，并且与传统的植物提取或化学合成相比，生物转化方法产生的环境污染更少，并具有可持续生产高茴香油的巨大潜力。ROMT-9 0.16 g / L，SAM 0.2 g / L，CH 3 OH 6％（v / v），温度37°C，pH 8.0。这项工作提供了一种高效合成高茴香油的替代策略，与传统的植物提取或化学合成相比，生物转化方法产生的环境污染更少，并且具有可持续生产高茴香油的巨大潜力。ROMT-9 0.16 g / L，SAM 0.2 g / L，CH 3 OH 6％（v / v），温度37°C，pH 8.0。这项工作提供了一种高效合成高茴香油的替代策略，与传统的植物提取或化学合成相比，生物转化方法产生的环境污染更少，并且具有可持续生产高茴香油的巨大潜力。