Metabolic engineering of the ethylmalonyl-CoA pathway in M. extorquens AM1 for 1-butanol production
<italic>M. extorquens</italic> AM1 has several advantages as a potential platform strain for industrial production of valuable chemicals, such as the capability of using an inexpensive carbon feedstock, clarified central metabolism, and availability of genetic tools. However, several fundamental questions involving how metabolic pathways are regulated and how they function as a system remained unsolved, which impedes progress in the metabolic engineering of <italic>M. extorquens</italic> AM1. This work is a compilation of efforts aiming to address initial issues related to the metabolic engineering of <italic>M. extorquens</italic> AM1 and establish a synthetic 1-butanol pathway as a proof of principle case. First, we investigated and characterized the effect of a tetR-type regulator (CcrR) on the expression of ccr, a critical gene of the EMC pathway, a key target for metabolic engineering. Furthermore, to demonstrate the potential of <italic>M. extorquens</italic> AM1 as a future industrial microorganism, a 1-butanol synthesis pathway was established in M. extorquens AM1 by diverting part of the native crotonyl-CoA flux from the EMC pathway to 1-butanol production. Engineered strains demonstrated different 1-butanol production using ethylamine as a substrate, and optimization was carried out. Finally, adaptive laboratory evolution was used as a tool to develop <italic>M. extorquens</italic> AM1 for high 1-butanol tolerance. We applied a serial transfer method to isolate two mutants that demonstrated improved 1-butanol tolerance compared to the parent strain. Whole genome sequencing of evolved strains revealed mutations that may be responsible for the observed phenotype. Our success in producing 1-butanol from the engineered <italic>M. extorquens</italic> AM1 opens the possibility for using methylotrophs for bulk chemical production. In addition, the collected information from this research will provide useful guidance for metabolic engineering of <italic>M. extorquens</italic> AM1 as an industrial platform in the future.
- Chemical engineering