切换格式脱酶的辅因子偏好,以开发一种依赖NADPH的生物催化系统,用于合成基拉尔氨基酸
Feng Cheng1,2, Lan Wei1,2, Cheng-Jiao Wang1,2
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China.
Journal of agricultural and food chemistry
|June 2, 2023
概括
工程化形式脱酶 (FDH) 酶现在有效地再生NADP,克服了以前的限制. 这一突破使得新的生物催化应用成为可能,包括l-phosphinothricin的合成.
科学领域:
- 生物催化和酶工程 生物催化和酶工程
- 生物化学工程 生物化学工程
- 合成生物学 合成生物学
背景情况:
- 基于甲酸脱酶 (FDH) 的辅因子再生对于生物催化剂至关重要.
- 现有的FDHs主要再生NAD,这对NADPH再生构成挑战.
- 活动特异性的权衡往往限制了酶工程的努力.
研究的目的:
- 来自*Azospirillum palustre* (*Ap*FDH) 的形式脱酶 (FDH) 进行工程,以实现高效的NADP+再生.
- 为了克服FDH工程中的活动特异性-稳定性权衡.
- 开发和应用一个工程化的FDH用于l-phosphinothricin的不对称生物合成.
主要方法:
- 选择一个高活动的*Ap*FDH.
- 位点定向突变发生 (D222Q) 来切换辅因子偏好.
- 半导体图书馆设计和选,以识别有益突变 (A199G,H380S).
- 结构和动态分析以了解突变效应.
- 在NADPH再生系统中工程FDH的应用,用于l-phosphinothricin合成.
主要成果:
- 一个单一的D222Q突变改变了辅因子偏好,但其活性降低了90%.
- 三重突变*Ap*FDHD222Q+A199G+H380S表现出改善的活动和NADP+的特异性.
- 一个进一步优化的变种 (D222Q-A199G/H380S-C256A/C146S) 同时增强了活动,特异性和稳定性.
- 设计的*Ap*FDH已成功应用于其他17个FDH.
- 使用工程化*Ap*FDH开发了一个优化的NADPH再生系统.
结论:
- 工程*Ap*FDH变体成功克服了NADP+再生的活性特异性稳定性权衡.
- 开发的酶工程策略广泛适用于其他FDHs.
- 设计的FDH系统使得l-phosphinothricin的高效不对称生物合成成为可能,证明了其实际的生物催化效用.
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