5-10: Engineering ethylmalonyl-CoA pathway in Methylobacterium extorquens AM1 for butanol production: identification of a regulator activating the expression of a key gene

As the best understood methylotroph, Methylobacterium extorquens AM1 is a potential platform for converting methanol to biofuels after the elucidation of pathways involved in C1 and C2 metabolism and development of tools for metabolic engineering. The ethylmalonyl-CoA pathway, one of the central methylotrophy pathways in this bacterium, involves several intermediates of biotechnology interest that can potentially be withdrawn for production of valuable chemicals. Therefore, understanding of the regulatory mechanism of fluxes of these intermediates, with special emphasis on crotonyl-CoA, which is a precursor for butanol synthesis, is an active area of research.

In this work, CcrR, a TetR-type activator, has been shown to regulate the expression of crotonyl-CoA reductase/carboxylase. The ccrR mutant is impaired in its ability to grow on C1 and C2 compounds, correlating with the reduced activity of crotonyl-CoA reductase/carboxylase. The ccr gene was found to be cotranscribed with an upstream gene (katA) and the experimental results show that CcrR stimulates expression of the katA-ccr promoter on the order of two-fold but is not required for this expression. A palindrome sequence upstream of katA at position -334 to -321 with respect to the translational start site was found to be the binding site. This work is not only the first step in understanding the regulation mechanism of crotonyl-CoA, but also generates information useful for engineering a butanol synthesis pathway using crotonyl-CoA as precursor.