香芹醇是一种具有重要医药、食品及日化应用价值的单萜类化合物,通常通过微生物或其酶催化柠檬烯氧化获得。然而,目前报道的氧化酶活性较低且专一性不足,难以满足香芹醇大规模工业生产的需求。因此,该研究以具有催化柠檬烯生成香芹醇能力的克雷伯杆菌O852为研究对象,基于前期获得的基因组数据,通过本地BLAST和生物信息学分析,结合多序列比对及进化树分析,挖掘克雷伯杆菌O852中催化柠檬烯生成香芹醇的关键酶,即包含多组分酶系统的Rieske型芳香环双加氧酶(末端氧化酶KlebDOX1和铁氧还蛋白/铁氧还蛋白还原酶KlebFdR1、末端氧化酶KlebDOX2和铁氧还蛋白/铁氧还蛋白还原酶KlebFdR2)。为了进一步验证这些酶的功能,对其基因进行克隆和重组表达,并通过柠檬烯氧化实验证实双加氧酶系KlebDOX1-KlebFdR1和KlebDOX2-KlebFdR2能够催化柠檬烯氧化生成香芹醇。该研究获得了可用于合成香芹醇的关键酶,丰富了酶资源库,为香芹醇的生物合成奠定了坚实的基础。
Carveol is a monoterpene compound with significant values in the fields of medicine, food, and daily chemicals.It is usually obtained through the oxidation of limonene catalyzed by microorganisms or their enzymes.Nevertheless, the enzyme activity reported at present is relatively low and its specificity is inadequate, which makes it challenging to meet the requirements for large-scale industrial production of carveol.In this study, Klebsiella O852, which had the capacity to catalyze the conversion of limonene to carveol, was selected as the research subject.Based on the previously acquired genomic data, through local BLAST and bioinformatics analyses, in combination with multiple sequence alignment and phylogenetic tree analyses, the key enzymes that catalyzed the generation of carveol from limonene in Klebsiella sp.O852 were identified, namely the multi-component enzyme system of Rieske-type aromatic ring dioxygenase (terminal oxidase KlebDOX1 and ferredoxin/ferredoxin reductase KlebFdR1, as well as terminal oxidase KlebDOX2 and ferredoxin/ferredoxin reductase KlebFdR2).To further verify the functions of these enzymes, their genes were cloned and recombinantly expressed, and it was confirmed through limonene oxidation experiments that the dioxygenase systems KlebDOX1-KlebFdR1 and KlebDOX2-KlebFdR2 were capable of catalyzing the oxidation of limonene to generate carveol.This research has obtained the key enzymes applicable for the synthesis of carveol, enriched the enzyme resource pool, and laid a solid foundation for the biosynthesis of carveol.
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