Objectives

In this study, 44 flavone synthases (FNS) and flavonol synthases (FLS) from different origins were collected. The instability index and conserved domain of the enzymes were analyzed through bioinformatics analysis, the results of which allowed us to screen suitable enzymes for constructing recombinant Escherichia coli. Defective enzymes were selected as controls.

Results

Native- and sodium dodecyl sulfate–polyacrylamide gel electrophoresis were conducted to isolate the heterologously expressed proteins. Liquid chromatography-mass spectrometry, ¹H nuclear magnetic resonance, and ultra-performance liquid chromatography were performed to qualitatively and quantitatively analyze the products. The cellular transformation results showed that recombinant E. coli catalyzed the synthesis of diosmetin from hesperetin, and in vitro catalysis showed that heterologously expressed FNS/FLS played a catalytic role in this reaction. AnFNS (from Angelica archangelica) showed the highest substrate conversion (38.80% for cellular transformation, 12.93% for in vitro catalysis).

Conclusions

The catalytic capacity of FNS/FLS from different origins exhibited the expected results, indicating that bioinformatics analysis is useful for screening enzymes. In addition, the catalytic properties of AnFNS and CaFLS (from Camellia sinensis) differed significantly, although these enzymes are structurally similar. Based on this difference, C-2 was predicted as the key site for FNS/FLS catalytic synthesis of diosmetin rather than C-3.

中文翻译：

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黄酮合酶和黄酮醇合酶催化橙皮素生成薯蓣素的筛选及异源表达

目标

在这项研究中，收集了来自不同来源的 44 种黄酮合酶 (FNS) 和黄酮醇合酶 (FLS)。通过生物信息学分析分析了酶的不稳定性指数和保守结构域，其结果使我们能够筛选出适合构建重组大肠杆菌的酶。选择有缺陷的酶作为对照。

结果

进行天然和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以分离异源表达的蛋白质。采用液相色谱-质谱、¹ H核磁共振和超高效液相色谱对产物进行定性和定量分析。细胞转化结果表明，重组大肠杆菌催化橙皮素合成薯蓣素，体外催化表明异源表达的FNS/FLS对该反应起催化作用。AnFNS（来自Angelica archangelica）显示出最高的底物转化率（细胞转化为 38.80%，体外催化为 12.93%）。

结论

不同来源的 FNS/FLS 的催化能力表现出预期的结果，表明生物信息学分析可用于筛选酶。此外，AnFNS 和 CaFLS（来自Camellia sinensis）的催化特性显着不同，尽管这些酶在结构上相似。基于这种差异，C-2 被预测为 FNS/FLS 催化合成薯蓣皂苷的关键位点，而不是 C-3。