Multiple Formate Dehydrogenase Enzymes in the Facultative Methylotroph Methylobacterium extorquens AM1 Are Dispensable for Growth on Methanol

Abstract

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C 1 compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.The classic scheme of energy metabolism during methylotrophic growth involves a formate oxidation step except in strains in which all formaldehyde is oxidized in the cyclic ribulose monophosphate cycle (1). Formate dehydrogenase (FDH) activity has been detected in most methylotrophs (3,10,13,15,22,42), and a few FDHs have been purified and analyzed (reviewed in reference 39). In the methylotrophic yeast Candida boidinii, the FDH step was shown not to be essential for methylotrophic growth, but FDH mutants showed reduced growth on methanol (32). However, as the complete C. boidinii genome sequence is not available, the presence of other FDHs is not excluded.Mutant-based analysis of the role of the FDH step in C 1 oxidation has not yet been attempted in methylotrophic bacteria. M. extorquens AM1 offers a convenient model to study this question. It possesses two pathways in which formaldehyde can be oxidized to formate (Fig. 1), one linked to tetrahydromethanopterin (H 4 MPT) and another linked to tetrahydrofolate (H 4 F) (5, 6). The enzymes involved in the two pathways have been studied in detail, and current evidence suggests that the main pathway for oxidizing formaldehyde is the H 4 MPTlinked pathway (reviewed in reference 37). It has been demonstrated recently that this pathway produces formate as an intermediate, a result of a formylmethanofuran transferase/ hydrolase reaction (29), and thus in this pathway one molecule of formate is formed in M. extorquens AM1 per oxidized molecule of a C 1 substrate, such as methanol or methylamine. This formate is subsequently oxidized to CO 2 , presumably by FDH (Fig. 1).An FDH from M. extorquens AM1 has recently been purified and characterized and shown to be a novel, tungsten-containing FDH encoded by two genes, fdh1AB (20). In this study we identified two new regions in the M. extorquens AM1 chromosome coding for two addition...

Genomic Analysis of Carbon Source Metabolism of Shewanella oneidensis MR-1: Predictions versus Experiments

“…Otherwise Shewanella spp. could employ an as yet unknown pathway as a recycling mode for capturing carbon when growing on formate or formaldehyde as a carbon source (5). Biochemical investigation is needed.…”

OptStrain: A computational framework for redesign of microbial production systems

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

“…Formate is an important branch point for the distribution between assimilation and energy production (11). (iii) Part of the formate pool is further oxidized to CO 2 due to the activity of formate dehydrogenases (12,13). (iv) The remainder of the formate pool undergoes a series of enzymatic steps catalyzing the conversion of formate to methylene-tetrahydrofolate (methylene-H 4 F), the intermediate that is incorporated into the assimilation cycles.…”