Alternative route for glyoxylate consumption during growth on two-carbon compounds by Methylobacterium extorquens AM1

Abstract

Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C(1) and C(2) compounds, showing that both modes of growth have this pathway in common. However, growth on C(2) compounds via the ethylmalonyl-CoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/beta-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C(2) compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and (13)C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/beta-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C(2) compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H(4)F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C(2) compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.

FIG. 1.

Enzymes and genes involved in the ethylmalonyol-CoA pathway. The colors of gene names denote a change in gene expression from microarray results comparing wild-type cells grown on ethylamine to those grown on succinate: dark red, >3-fold increase; light red, 1.5- to 3-fold increase; black, no significant change (1.49-fold increase to 1.49-fold decrease); light green, 1.5- to 3-fold decrease; dark green, >3-fold decrease. Parentheses denote a predicted function not confirmed by the mutant phenotype. See Table 1 for enzyme names.

FIG. 2.

Potential pathway for converting glyoxylate to 2-phosphoglycerate via glycine and serine. The net reaction is shown at the bottom. Gene name colors are the same as those describe for Fig. 1.

FIG. 3.

Possible fates of glyoxylate in M. extorquens AM1. Parentheses denote a predicted function not confirmed by the mutant phenotype. See Table 1 for enzyme names.

FIG. 4.

Comparison of intracellular and supernatant metabolites in wild-type and mutant strains grown on ethylamine. Results are ratios of values from each mutant compared to wild-type values. Top, supernatant; bottom, intracellular. *, significant difference (P < 0.05) between mutants and the wild type. Error bars show the range for three biological replicates, each with three technical replicates. 3Mt2oxoPen, 3-methyl-2-oxopentanoic acid; 4Mt2oxoPen, 4-methyl-2-oxopentanoic acid; MtSuc, methylsuccinic acid; Pyru, pyruvate.

FIG. 5.

Predicted labeling patterns from [¹³C]glyoxylate. Unlabeled products are not shown.

FIG. 6.

Time course of ratios of isotopomers of glycine (top), serine (middle), and malate (bottom) after the incubation of ethylamine-grown cells with [¹³C]glyoxylate in the presence of [¹²C]ethylamine as a fraction of the total compound. M + 0, unlabeled; M + 1, singly labeled; M + 2, doubly labeled; M + 3, triply labeled; M + 4, quadruply labeled.