Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using ¹³C‐label tracing and mass spectrometry

Abstract

The metabolic fluxes of central carbon metabolism were measured in chemostat-grown cultures of Methylobacterium extorquens AM1 with methanol as the sole organic carbon and energy source and growth-limiting substrate. Label tracing experiments were carried out using 70% (13)C-methanol in the feed, and the steady-state mass isotopomer distributions of amino acids derived from total cell protein were measured by gas chromatography coupled to mass spectrometry. Fluxes were calculated from the isotopomer distribution data using an isotopomer balance model and evolutionary error minimization algorithm. The combination of labeled methanol with unlabeled CO(2), which enters central metabolism in two different reactions, provided the discriminatory power necessary to allow quantification of the unknown fluxes within a reasonably small confidence interval. In wild-type M. extorquens AM1, no measurable flux was detected through pyruvate dehydrogenase or malic enzyme, and very little flux through alpha-ketoglutarate dehydrogenase (1.4% of total carbon). In contrast, the alpha-ketoglutarate dehydrogenase flux was 25.5% of total carbon in the regulatory mutant strain phaR, while the pyruvate dehydrogenase and malic enzyme fluxes remained insignificant. The success of this technique with growth on C(1) compounds suggests that it can be applied to help characterize the effects of other regulatory mutations, and serve as a diagnostic tool in the metabolic engineering of methylotrophic bacteria.

Increasing the accuracy of mass isotopomer analysis through calibration curves constructed using biologically synthesized compounds

“…This step was designed to avoid dilution of isotope abundance caused by assimilation of naturally CO 2 via carboxylation reactions of the bacteria. [43] After autoclave, 13 C-labeled methanol was added in the medium to a final concentration of 1% (v/v). Frozen stock of M. salsuginis sp.…”

Identification of a Fourth Formate Dehydrogenase in Methylobacterium extorquens AM1 and Confirmation of the Essential Role of Formate Oxidation in Methylotrophy

“…The following antibiotic concentrations were used: kanamycin, 100 g/ml; rifamycin, 50 g/ml; and tetracycline, 10 g/ml. To test the metal-dependent expression of FDHs, a similar medium was used in which molybdenum was omitted and molybdenum, tungsten, or both were added, as appropriate, at 0.3 to 3 M. Chemostat cultivation was performed in a bench-top fermentor essentially as previously described (5,21). The methanol concentration in the feed was 25 mM for the wild type and quadruple mutant A and either 25 or 45 mM for the triple-FDH mutant, and dilution rates were 0.1 h Ϫ1 unless otherwise stated.…”

OptStrain: A computational framework for redesign of microbial production systems

“…The organism has been well studied (Anthony 1982;Chistoserdova et al 1998;Korotkova et al 2002;Van Dien et al 2003;Chistoserdova et al 2004), and recently, a stoichiometric model of its central metabolism was published (Van Dien and Lidstrom 2002). Using Step 3 of OptStrain, we identified that only a single reaction needs to be introduced into the metabolic network of M. extorquens to enable hydrogen production.…”