Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis

Abstract

The growth of Methylobacterium extorquens AM1 on C 1 compounds has been well-studied, but little is known about how this methylotroph grows on multicarbon compounds. A Tn5 transposon mutagenesis procedure was performed to identify genes involved in the growth of M. extorquens AM1 on succinate and pyruvate. Of the 15 000 insertion colonies screened, 71 mutants were found that grew on methanol but either grew slowly or were unable to grow on one or both of the multicarbon substrates. For each of these mutants, the chromosomal region adjacent to the insertion site was sequenced, and 55 different genes were identified and assigned putative functions. These genes fell into a number of predicted categories, including central carbon metabolism, carbohydrate metabolism, regulation, transport and non-essential housekeeping functions. This study focused on genes predicted to encode enzymes of central heterotrophic metabolism: 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase and NADH : ubiquinone oxidoreductase. In each case, the mutants showed normal growth on methanol and impaired growth on pyruvate and succinate, consistent with a role specific to heterotrophic metabolism. For the first two cases, no detectable activity of the corresponding enzyme was found in the mutant, verifying the predictions. The results of this study were used to reconstruct multicarbon metabolism of M. extorquens AM1 during growth on methanol, succinate and pyruvate.

Methylotrophy in Methylobacterium extorquens AM1 from a Genomic Point of View

“…This result points toward either succinyl-CoA hydrolase being the essential enzyme for this conversion or the three systems being degenerate for this function. The genes for succinate dehydrogenase (SDH) are all linked on the chromosome (cluster 29), and SDH null mutants cannot be obtained either on methanol or on succinate (48,49). However, they can be obtained on methanol supplemented with glyoxylate (N. Korotkova and M. E. Lidstrom, unpublished observations).…”

“…In contrast, it is not involved in energy generation during growth on C 1 compounds. Instead, ␣-ketoglutarate dehydrogenase is repressed, and the incomplete cycle plays a strictly assimilatory role (3,48,49). However, many of the TCA cycle enzymes are involved in C 1 assimilation.…”

Methenyl-Dephosphotetrahydromethanopterin Is a Regulatory Signal for Acclimation to Changes in Substrate Availability in Methylobacterium extorquens AM1

“…Methanol metabolism is predicted to be limited by reducing power (1-3), requires the cofactor dephosphotetrahydromethanopterin (dH 4 MPT) (4, 5), uses the serine cycle for assimilation, and involves high carbon flux through toxic intermediates, such as formaldehyde, glyoxylate, and glycine (6). In contrast, multicarbon growth is predicted to be energy limited and involves common heterotrophic pathways, such as the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway (7,8).Methanol and methylamine metabolism can be divided into five functional metabolic steps, as follows. (i) The oxidation of methanol or methylamine to formaldehyde is catalyzed by either methanol dehydrogenase (MeDH) or methylamine dehydrogenase, respectively.…”

Introducing a new Bioengineered Bug:Methylobacterium extorquenstuned as a microbial bioplastic factory

“…Knowing that M. extorquens is incapable of growing on multicarbon substrates (C > 4), research has focused so far on metabolic behavior and fluxes resulting from C1-C4 components. 8,20,21 In this article, we discuss the metabolic insights we have obtained from exposing wild-type and recombinant M. extorquens ATCC 55366 strains to some MCL alkenoic acids (C > 5).…”