Fan Liwen, Wang Yu, Zheng Ping, Sun Jibin
School of Life Sciences, University of Science and Technology of China, Hefei 230026, Anhui, China.
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Feb 25;37(2):530-540. doi: 10.13345/j.cjb.200335.
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.
甲醇和甲烷等一碳化合物是生物制造中廉价且易于获取的原料。甲醇脱氢酶（MDH）催化甲醇氧化为甲醛是微生物一碳代谢的关键步骤。从天然甲基营养菌中发现了多种依赖不同辅因子且具有不同酶学性质的MDH。烟酰胺腺嘌呤二核苷酸（NAD）依赖性MDH被广泛用于构建合成甲基营养菌，然而这类MDH通常甲醇氧化活性较低且对甲醇的亲和力较低。因此，甲醇氧化被认为是合成甲基营养菌中甲醇代谢的限速步骤。为了加速甲醇氧化，从而提高合成甲基营养菌对甲醇的利用效率，大量研究聚焦于MDH的发现与工程改造。在本综述中，我们总结了在发现、表征和改造各类MDH方面所做的努力，以及MDH在合成甲基营养菌中的应用。详细描述了MDH的定向进化和多酶复合物的构建。在未来展望部分，我们讨论了通过生长偶联蛋白进化和合理蛋白设计来获得优质MDH的潜在策略。
