Protein engineering of nicotinamide coenzyme-dependent oxidoreductases for coenzyme preference and its application in synthetic biology

doi:10.12211/2096-8280.2020-038

Abstract

Abstract:

Nicotinamide-based coenzyme NAD(P) is the most common electron mediator in the redox processes in living organisms. As NAD and NADP play an important role in catabolism and anabolism, it is essential to ensure their supply and consumption, as well as to maintain their balance in synthetic biological systems. Synthetic pathways that fail to match coenzyme supply with demand will probably result in low product yield and slow volumetric productivity. To solve the problem of coenzyme imbalance, the best strategy is to change the coenzyme preference of oxidoreductases in the pathway by protein engineering and then replace the wild-type enzymes with the mutants. In addition, biomimetic coenzymes can be designed and used to replace natural nicotinamide-based coenzymes for redox reactions due to the low cost and better stability. However, most of the oxidoreductases in nature have no or little activity on biomimetic coenzymes. Therefore, this review first focuses on the methodology and research progress of natural nicotinamide-based coenzyme engineering, with its application in improving product yield and decreasing production cost. Studies on the utilization of protein engineering technology for the switching of coenzyme preference from natural to biomimetic coenzymes are also presented, indicating a broad application prospect of biomimetic coenzymes in the construction of both in vivo bioorthogonal redox pathways and in vitro synthetic enzymatic pathways. Despite some general rules have been proposed for natural coenzyme engineering, coenzyme engineering for changing the preference of oxidoreductases on biomimetic coenzymes remains its early stage due to the significant differences in structures and sizes among various natural and biomimetic coenzymes. Nevertheless, with the increasing numbers of resolved high-resolution protein crystal structures and homogeneous oxidoreductase sequences, the development of novel high-throughput screening methods, as well as the design of more biomimetic coenzymes with improved properties, the modification of coenzyme preference from natural to biomimetic coenzymes will become a prior direction of coenzyme engineering in the future.

Key words: electron mediator, protein engineering, biomimetic coenzymes, natural coenzymes, synthetic biology

摘要：

烟酰胺类辅酶NAD（P）是生命体氧化还原过程中最常见的电子中介体。绝大多数氧化还原酶都需要NAD或NADP进行分解代谢和合成代谢，但根据其在代谢过程中的作用，不同氧化还原酶表现出不同的辅酶偏好性。为了解决辅酶在代谢过程中的供需平衡、生产成本和稳定性等问题，对氧化还原酶的辅酶偏好性改造是蛋白质工程的研究热点。本文综述了氧化还原酶的天然烟酰胺辅酶偏好性的改造方法和研究进展，以及天然辅酶偏好性改造工程在提高产品收率和降低生产成本方面的应用。仿生烟酰胺辅酶因成本低、稳定性高，常被用来代替天然烟酰胺辅酶参与氧化还原反应，因此本文还详细介绍了改造酶元件使其偏好仿生烟酰胺辅酶以及其在合成生物学领域构建体内生物正交氧化还原途径和体外合成途径等方面的研究进展。随着更多天然酶元件以及新型优良仿生辅酶的挖掘和设计，从天然辅酶到仿生辅酶的辅酶偏好性改造已成为辅酶工程的一个重要方向。

关键词: 电子中介体, 蛋白质工程, 仿生烟酰胺辅酶, 天然烟酰胺辅酶, 合成生物学

CLC Number:

Q 599

Meixia LIU, Qiangzi LI, Dongdong MENG, Xinlei WEI, Chun YOU. Protein engineering of nicotinamide coenzyme-dependent oxidoreductases for coenzyme preference and its application in synthetic biology[J]. Synthetic Biology Journal, 2020, 1(5): 570-582.

刘美霞, 李强子, 孟冬冬, 魏欣蕾, 游淳. 烟酰胺类辅酶依赖型氧化还原酶的辅酶偏好性改造及其在合成生物学中的应用[J]. 合成生物学, 2020, 1(5): 570-582.

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