一碳代谢关键酶——甲醇脱氢酶的研究进展与展望

Methanol dehydrogenase, a key enzyme of one-carbon metabolism: a review

摘要:

甲醇和甲烷等一碳原料来源广泛，价格低廉，是生物制造的理想原料。甲醇脱氢酶 (Methanol dehydrogenase，MDH) 催化甲醇生成甲醛是一碳代谢的关键反应。目前已从天然甲基营养菌中发现了多种利用不同辅因子，具有不同酶学性质的MDH。其中，烟酰胺腺嘌呤双核苷酸 (NAD) 依赖型MDH被广泛应用于构建人工甲基营养菌。但是，NAD依赖型MDH的甲醇氧化活性较低，对甲醇的亲和力较差，导致甲醇氧化成为人工甲基营养菌代谢甲醇的限速步骤。为了提高甲醇氧化速率，进而提高人工甲基营养菌的甲醇利用效率，近年来大量研究集中于MDH的挖掘与改造研究。文中系统综述了不同类型MDH的发现、表征、改造以及在人工甲基营养菌中的应用进展，详细阐述了MDH的定向进化和多酶复合体的构建，并展望了通过细胞生长偶联的蛋白质进化和蛋白质理性设计获得高活性MDH的潜在策略。

Abstract:

One-carbon compounds such as methanol and methane are cheap and readily available feedstocks for biomanufacturing. Oxidation of methanol to formaldehyde catalyzed by methanol dehydrogenase (MDH) is a key step of microbial one-carbon metabolism. A variety of MDHs that depend on different co-factors and possess different enzymatic properties have been discovered from native methylotrophs. Nicotinamide adenine dinucleotide (NAD)-dependent MDHs are widely used in constructing synthetic methylotrophs, whereas this type of MDH usually suffers from low methanol oxidation activity and low affinity to methanol. Consequently, methanol oxidation is considered as a rate-limiting step of methanol metabolism in synthetic methylotrophs. To accelerate methanol oxidation, thereby improving the methanol utilization efficiency of synthetic methylotrophs, massive researches have focused on discovery and engineering of MDHs. In this review, we summarize the ongoing efforts to discover, characterize, and engineer various types of MDHs as well as the applications of MDHs in synthetic methylotrophs. Directed evolution of MDH and construction of multi-enzyme complexes are described in detail. In the future prospective part, we discuss the potential strategies of growth-coupled protein evolution and rational protein design for acquisition of superior MDHs.