Our work involving Methylobacterium extorquens AM1 falls into 3 major classes, fundamental physiological studies, regulatory studies, and metabolic engineering. All of this work takes advantage of the recently completed genomic sequence. The long-term goal of these projects is to use parallel techniques to gain a systems-level understanding of this organism and to use that to manipulate methylotrophic metabolism for commercial processes.
The goal of this work is to understand the metabolic pathways of the various parts of methylotrophy and how they interconnect with central metabolism. These studies involve identification of genes that may be involved in methylotrophy, mutation of key genes by insertional mutagenesis, and analysis of mutant phenotypes. So far we have identified approximately 100 genes that are involved in methylotrophy. Many of these are organized in operons and large, multi-operon gene clusters (‘methylotrophy islands’).
Recently, we have generated whole genome cDNA microarrays and are using them to investigate genes and metabolic pathways involved in C1 and C2 metabolism, formaldehyde stress response, growth rate regulation and methylotrophic regulatory networks. These microarray studies along with proteomic studies done in collaboration with M. Hackett et al, have identified a number of new candidate genes that may have a role in methylotrophic metabolism including a forth formate dehydrogenase that is responsive to C1 metabolism and acid stress and operon containing genes involved in formate metabolism. Candidate genes have been mutated and phenotypic and biochemical studies are underway to define the areas of methylotrophy altered in these mutants.
The goal of this work is to understand the molecular basis of expression and regulation of methylotrophic processes and the identification of new regulatory networks. These studies involve identification of promoters and regulatory genes, analysis by deletion and mutation, and transcript analysis. Our data so far suggest that methanol-regulated promoters are similar to poor E. coli s70 promoters, enhanced by nearby upstream positive regulatory elements. Formaldehyde is apparently a key regulatory effector along with formate and formyl-H4F. Currently, we are investigating the interconnection between methylotropic, iron and QscR-mediated regulation. QscR is an autorepressor and a transcriptional activator of the serine cycle genes. Microarray, promoter fusion and gel retardation studies suggest that QscR is a global regulator responsible for the activation and repression of numerous genes outside of methylotrophic metabolism.
Both steady-state and kinetic models are being developed for methylotrophy, to generate predictions regarding the importance of specific pathway branches to parameters such as growth rate and biomass production. In addition, we are targeting specific natural products for overproduction, as model systems for metabolic engineering to convert methanol into value added products. These predictions are being tested by adding, deleting, or altering genes, as appropriate and then the modified strains are subjected to multi-tiered analysis involving microarrays, proteomics, metabolomics, and flux measurements. This then leads to an iterative improvement cycle.