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Background  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.    Results  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.    Conclusions  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.

Metabolic engineering of Methylobacterium extorquens AM1 for 1-butanol production