Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstrasse 1, Düsseldorf, 40225, Germany.
Chembiochem. 2022 Jun 20;23(12):e202200065. doi: 10.1002/cbic.202200065. Epub 2022 Apr 6.
Multi-enzyme cascades enable the production of valuable chemical compounds, and fusion of the enzymes that catalyze these reactions can improve the reaction outcome. In this work, P450 BM3 from Bacillus megaterium and an alcohol dehydrogenase from Sphingomonas yanoikuyae were fused to bifunctional constructs to enable cofactor regeneration and improve the in vitro two-step oxidation of (+)-valencene to (+)-nootkatone. An up to 1.5-fold increased activity of P450 BM3 was achieved with the fusion constructs compared to the individual enzyme. Conversion of (+)-valencene coupled to cofactor regeneration and performed in the presence of the solubilizing agent cyclodextrin resulted in up to 1080 mg L (+)-nootkatone produced by the fusion constructs as opposed to 620 mg L produced by a mixture of the separate enzymes. Thus, a two-step (+)-valencene oxidation was considerably improved through the simple method of enzyme fusion.
多酶级联反应能够生成有价值的化合物，而融合催化这些反应的酶可以改善反应结果。在这项工作中，来自巨大芽孢杆菌的 P450 BM3 和来自粘质沙雷氏菌的醇脱氢酶被融合到双功能构建体中，以实现辅因子再生，并提高 (+)-柠檬烯向 (+)-诺卡酮的体外两步氧化。与单独的酶相比，融合构建体实现了 P450 BM3 活性提高了 1.5 倍。在增溶剂环糊精存在下，与辅因子再生偶联的 (+)-柠檬烯的转化导致融合构建体产生的 (+)-诺卡酮高达 1080mg/L，而单独的酶混合物产生的 (+)-诺卡酮为 620mg/L。因此，通过简单的酶融合方法，(+)-柠檬烯的两步氧化得到了显著改善。
