Daidzein is a major isoflavone compound with an immense pharmaceutical value. This study applied a novel P450 CYP82D26 which can biosynthesize daidzein from (2S)-naringenin. However, the recombinant P450 systems often suffer from low coupling efficiency, leading to an electron transfer efficiency decrease and harmful reactive oxygen species release, thereby compromising their stability and catalytic efficiency. To address these challenges, the SH3-GBD-PDZ (SGP) protein scaffold was applied to assemble a multienzyme system comprising CYP82D26, P450 reductase, and NADP⁺-dependent aldehyde reductase in desired stoichiometric ratios. Results showed that the coupling efficiency of the P450 system was significantly increased, primarily attributed to the channeling effect of NADPH resulting from the proximity of tethered enzymes and the electrostatic interactions between NADPH and SGP. Assembling this SGP-scaffolded assembly system in Escherichia coli yielded a titer of 240.5 mg/L daidzein with an 86% (2S)-naringenin conversion rate, which showed a 9-fold increase over the free enzymes of the P450 system. These results underscore the potential application of the SGP-scaffolded multienzyme system in enhancing the coupling and catalytic efficiency of the P450 system.

中文翻译：

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构建蛋白质支架多酶组装提高 P450 系统的偶联效率，以从 (2S)-柚皮素高效生物合成黄豆苷元

大豆黄酮是一种主要的异黄酮化合物，具有巨大的药用价值。本研究应用了一种新型P450 CYP82D26，它可以从(2S)-柚皮素生物合成大豆黄素。然而，重组P450系统往往存在偶联效率低的问题，导致电子转移效率降低和有害活性氧释放，从而损害其稳定性和催化效率。为了应对这些挑战，应用 SH3-GBD-PDZ (SGP) 蛋白支架组装多酶系统，该系统包含所需化学计量比的 CYP82D26、P450 还原酶和 NADP ⁺依赖性醛还原酶。结果表明，P450 系统的偶联效率显着提高，这主要归因于束缚酶的接近以及 NADPH 和 SGP 之间的静电相互作用所产生的 NADPH 通道效应。在大肠杆菌中组装这种 SGP 支架组装系统，产生了 240.5 mg/L 的黄豆苷元滴度和 86% (2 S )-柚皮素转化率，比 P450 系统的游离酶增加了 9 倍。这些结果强调了SGP支架多酶系统在增强P450系统的偶联和催化效率方面的潜在应用。