Metabolic control of the ethylmalonyl-CoA pathway in Methylobacterium extorquens AM1

Abstract

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on reduced carbon compounds with no carbon-carbon bonds, as well as multi-carbon compounds. M. extorquens AM1 is widespread in nature and is often associated with the plant phyllosphere, a heterogeneous environment with consistently changing substrate availability. During growth on one-carbon substrates, such as the renewable feedstock methanol, M. extorquens AM1 utilizes the ethylmalonyl-CoA pathway that involves several rare four- and five carbon CoA-esters of great biotechnological interest. M. extorquens AM1 also incorporates carbon dioxide as half of the fixed carbon during biomass production, making it a desirable organism to develop into a platform for the production of value-added chemicals. A better understanding of the regulation of the ethylmalonyl-CoA pathway is vital to future platform development of M. extorquens AM1. RNA-seq transcriptomics was developed and optimized for M. extorquens AM1. This method provided better replication and higher sensitivity than traditional microarrays for investigating gene expression, a primary level of regulation. With this approach validated, the ethylmalonyl-CoA pathway was investigated for a metabolic control point taking advantage of growth on ethylamine, a two-carbon substrate that is converted to biomass precursors through the ethylmalonyl-CoA pathway. Using a two-carbon substrate successfully bypassed identified regulatory mechanisms that restrict carbon flux into the assimilatory pathways during growth on methanol. The approach used involved challenging M. extorquens AM1 to switch from growth on succinate to growth on ethylamine and generating a timecourse of response measured by targeted metabolomics, RNA-seq transcriptomics, and enzyme activities. From these experiments, ethylmalonyl-CoA mutase was identified as a metabolic control point. To confirm the role of this enzyme as a metabolic control point, over-expression of ethylmalonyl-CoA mutase overrode the control and caused a change in growth phenotype. In this thesis, I demonstrated the utility of systems-level approaches to investigate a directed hypothesis and answer fundamental physiological questions, and have identified a candidate for future strain manipulation.