合成甲基营养细胞工厂同化甲醇的研究进展及未来展望

Research progresses and future prospects of synthetic methylotrophic cell factory for methanol assimilation

甲醇具有来源广、易储存运输、原料价格竞争力强等优势,被视为极具潜力的生物制造非糖基碳源资源。常用的模式底盘微生物研究历史长、认知清楚、操作工具多,在工程化改造中具有显著优势。近年来,通过借鉴天然甲基营养型微生物的甲醇利用途径对模式底盘进行改造,获得具备高效利用甲醇能力的合成甲基营养细胞工厂的研究日益受到关注。本文系统综述合成甲基营养细胞工厂的甲醇氧化、同化基因及其调控元件,和以共用糖基碳源结合适应性进化策略为主的核酮糖单磷酸途径重构及甲醇同化途径设计构建的研究现状,进而结合合成甲基营养细胞工厂面临的挑战进行展望,提出基于全基因组靶向基因编辑技术结合实验室适应性进化策略构建更高效合成甲基营养细胞工厂的研究方向。

Methanol is an important and attractive non-sugar carbon source for industry biotechnology due to its advantages of available source,easy storage,convenient transportation and competitive price.The widely-studied microorganisms such as Escherichia coli,Corynebacterium glutamicum and Saccharomyces cerevisiae have a long research history,clear genetic background and a number of matured genetic tools,showing their potential in engineering cell factories for bioproduction.With the accumulated knowledge of native methylotrophs in recent years,these traditional industrial microorganisms have been engineered as methylotrophic cell factories(MeCFs)capable of using methanol as the major carbon and energy source.In this artical,we review the recent research progress including genes involved in the methanol oxidation,assimilation and regulatory elements of MeCFs.We also summarized the strategies based on the adaptive laboratory evolution which applies sugar as the co-substrate to construct the ribulose monophosphate(RuMP)cycle,as well as strategies for designing and tuning methanol assimilation pathway.In the synthetic MeCFs,the construction of the RuMP cycle requires the introduction of three heterologous enzymes,i.e.,methanol dehydrogenase encoded by mdh,3-hexulose 6-phosphate synthase encoded by hps and 6-phospho 3-hexuloisomerase encoded by phi.These enzymes can be further engineered to improve activities through protein engineering and directed evolution.Meanwhile,genes involved in methanol oxidation and assimilation pathways can be finely tuned to exhibit dynamic expression.To further improve methanol utilization of the synthetic MeCFs,the cosubstrate for supporting growth has been developed to propel methanol assimilation,which rationally designs a methanol dependent growth model,and thus provides an ideal starting point for subsequent long-term adaptive laboratory evolution experiments.At the end,we discuss the challenges of engineering the synthetic MeCFs,and summarize the future prospects for improving the efficiency of methanol utilization through the combined strategies of genome-wide targeted gene editing and adaptive laboratory evolution.