K-104. New Insights into Methylotrophic Metabolism and Regulation in Methylobacterium extorquens AM1

E. Skovran, L. D. Palmer, M. E. Lidstrom;
Univ. of Washington, Seattle, WA.

Methylotrophic bacteria are of interest due to their environmental significance as well as their potential to be used in biocatalysis applications for the production of chemicals from renewable biofeedstocks such as methanol. Two approaches were used to find new genes required for methylotrophic growth and links to other metabolic pathways in Methylobacterium extorquens. First, global gene expression from multi-carbon chemostat grown cells (succinate) and single carbon chemostat grown cells (methanol and methylamine) was compared. Second, microarrays were used to follow gene expression after switching cells from succinate to methanol growth. Mutation of candidate genes resulted in 2 that were required for methylotrophic growth: a putative reductase and a gene of unknown function. Phenotypic and biochemical characterization showed that lesions in these genes have dramatic effects on the ability of M. extorquens to metabolize C1 compounds. The reductase clusters with 3 other genes forming a putative operon involved in formate metabolism. Loss of the reductase causes methanol sensitivity due to the accumulation of a toxic intermediate resulting in lowered formate dehydrogenase (Fdh) activity. Secondary mutations located in this cluster block the formation of this intermediate and allow growth on C1 compounds. A null mutation in the gene of unknown function has plieotrophic effects, resulting in excretion of formate (4.5 mM) and formaldehyde (0.3 mM). Loss of this gene also results in decreased activity of multiple C1 specific enzymes. Parallel studies showed that these defects are similar to those seen for strains lacking the C1 Serine Cycle transcriptional activator, QscR. Here we show that QscR also regulates several Fdhs and C1 transfer genes indicating that the decision to oxidize formate or convert it to biomass is a pivotal point of control during C1-growth. More studies are needed to determine whether loss of the gene of unknown function exerts its effects on methylotrophic metabolism through QscR, or is independent. Further characterization of these mutants will reveal much about methylotrophic metabolism and its regulation.