Metabolic engineering of Methylobacterium extorquens AM1 for 1-butanol production.
Metabolic engineering of Methylobacterium extorquens AM1 for 1-butanol production.
复制标题
1丁醇产生的甲基甲基甲基甲基甲虫的代谢工程。
DOI：
10.1186/s13068-014-0156-0
复制
发表时间：
2014
影响因子：
6.3
通讯作者：
Lidstrom ME
中科院分区：
文献类型：
--
作者：
Hu B;Lidstrom ME
丁醇是一种很有前途的新一代燃料，也是一种大宗化学前体。虽然梭状芽孢杆菌是发酵生产1-丁醇的主要工业微生物，但替代工程宿主有可能通过合成代谢途径从替代碳原料中生产1-丁醇。αextorquens AM1是一种兼性甲基营养的变形杆菌，是一种用于评估从一碳和两碳原料中生产正丁醇的可能性的模式系统。此外，Extorquens AM1的核心甲基营养途径涉及不寻常的辅酶A(CoA)衍生代谢物，如巴豆酰-CoA，它是生产1-丁醇的前体。在这项工作中，我们设计了一种改良的辅酶A依赖的途径，在甲基洛伯特氏菌AM1中产生1-丁醇。工程菌以乙胺为底物，表现出不同的正丁醇滴度。一株高效表达齿密螺旋体反式烯醇辅酶A还原酶、乙酰丁酸梭菌乙醇脱氢酶和天然巴豆酶的菌株，能产生最高的正丁醇效价(15.2mg.L−1)。体外代谢流同位素示踪和体内代谢物分析表明丁酰辅酶A积累，证明了合成途径的功能，并确定了未来改进的目标。我们以1-丁醇为例，论证了利用艾氏木霉AM1中乙基丙二酰辅酶A途径的代谢中间体生产增值化学品的可行性。这不仅将建立乙基丙二酰辅酶A途径的生物技术潜力，还将引入Extorquens AM1作为生产增值化学品的潜在平台。
Butanol is a promising next generation fuel and a bulk chemical precursor. Although clostridia are the primary industrial microbes for the fermentative production of 1-butanol, alternative engineered hosts have the potential to generate 1-butanol from alternative carbon feedstocks via synthetic metabolic pathways. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating products such as 1-butanol from one-carbon and two-carbon feedstocks. Moreover, the core methylotrophic pathways in M. extorquens AM1 involve unusual coenzyme A (CoA)-derivative metabolites, such as crotonyl-CoA, which is a precursor for the production of 1-butanol. In this work, we engineered a modified CoA-dependent pathway in Methylobacterium extorquens AM1 to produce 1-butanol. Engineered strains displayed different 1-butanol titers using ethylamine as a substrate. A strain overexpressing Treponema denticola trans-enoyl-CoA reductase, Clostridium acetobutylicum alcohol dehydrogenase, and native crotonase was able to generate the highest 1-butanol titer (15.2 mg l−1). In vitro isotopic tracing of metabolic flux and in vivo metabolite analysis showed the accumulation of butyryl-CoA, demonstrating the functionality of the synthetic pathway and identifying targets for future improvement. We demonstrated the feasibility of using metabolic intermediates of the ethylmalonyl-CoA pathway in M. extorquens AM1 to generate value-added chemicals, with 1-butanol as the test case. This will not only establish the biotechnological potential of the ethylmalonyl-CoA pathway, but will also introduce M. extorquens AM1 as a potential platform to produce value-added chemicals.