Methanol assimilation in Methylobacterium extorquens AM1: demonstration of all enzymes and their regulation

Abstract

Background: Methylobacterium extorquens AM1 is an aerobic facultative methylotrophic α-proteobacterium that can use reduced one-carbon compounds such as methanol, but also multi-carbon substrates like acetate (C₂) or succinate (C₄) as sole carbon and energy source. The organism has gained interest as future biotechnological production platform based on methanol as feedstock.

Methodology/principal findings: We present a comprehensive study of all postulated enzymes for the assimilation of methanol and their regulation in response to the carbon source. Formaldehyde, which is derived from methanol oxidation, is assimilated via the serine cycle, which starts with glyoxylate and forms acetyl-CoA. Acetyl-CoA is assimilated via the proposed ethylmalonyl-CoA pathway, which thereby regenerates glyoxylate. To further the understanding of the central carbon metabolism we identified and quantified all enzymes of the pathways involved in methanol assimilation. We observed a strict differential regulation of their activity level depending on whether C₁, C₂ or C₄ compounds are used. The enzymes, which are specifically required for the utilization of the individual substrates, were several-fold up-regulated and those not required were down-regulated. The enzymes of the ethylmalonyl-CoA pathway showed specific activities, which were higher than the calculated minimal values that can account for the observed growth rate. Yet, some enzymes of the serine cycle, notably its first and last enzymes serine hydroxymethyl transferase and malate thiokinase, exhibit much lower values and probably are rate limiting during methylotrophic growth. We identified the natural C₁ carrying coenzyme as tetrahydropteroyl-tetraglutamate rather than tetrahydrofolate.

Conclusion/significance: This study provides the first complete picture of the enzymes required for methanol assimilation, the regulation of their activity levels in response to the growth substrate, and the identification of potential growth limiting steps.

Figure 1. Scheme of C₁ metabolism of the methylotroph Methylobacterium extorquens AM1.

The bacterium oxidizes methanol to formaldehyde that is condensed with a tetrahydromethanopterin and further oxidized to formate. Formate reacts with tetrahydropterin and formyl-tetrahydrofolate is further converted to methylenetetrahydrofolate (part 1 of metabolism). The serine cycle is used for the assimilation of formaldehyde plus bicarbonate (part 2). Acetyl-CoA assimilation and conversion to glyoxylate proceeds via the ethylmalonyl-CoA pathway (part 3). The main biosynthetic outputs from these pathways are indicated. Enzymes: 1, serine hydroxymethyl transferase; 2, serine-glyoxylate aminotransferase; 3, hydroxypyruvate reductase; 4, glycerate kinase; 5, enolase; 6, phosphoenolpyruvate carboxylase; 7, malate dehydrogenase; 8, malate-CoA ligase (malate thiokinase); 9, L-malyl-CoA/β-methylmalyl-CoA lyase; 10, β-ketothiolase; 11, acetoacetyl-CoA reductase; 12, crotonase (R-specific); 13, crotonyl-CoA carboxylase reductase; 14, ethylmalonyl-CoA/methylmalonyl-CoA epimerase; 15, ethylmalonyl-CoA mutase; 16, methylsuccinyl-CoA dehydrogenase; 17, mesaconyl-CoA hydratase; 18, propionyl-CoA carboxylase; 19, methylmalonyl-CoA mutase; 20, methanol dehydrogenase. PHB, polyhydroxybutyrate, Q, quinone.

Figure 2. Specific activity of enzymes of the serine cycle and their regulation.

Enzymes: Mox, methanol dehydrogenase; GlyA, serine hydroxymethyl transferase; Sga, serine-glyoxylate aminotransferase; Hpr, hydroxypyruvate reductase; Gck, glycerate kinase; Eno, enolase; Ppc, phosphoenolpyruvate carboxylase; Mdh, malate dehydrogenase; Mtk, malate-CoA ligase; Mcl1, malyl-CoA/β-methylmalyl-CoA lyase. The y axis is in nmol min⁻¹ mg⁻¹ protein. M, methanol grown cells; A, acetate grown cells; S, succinate grown cells.

Figure 3. Specific activity of enzymes of the ethylmalonyl-CoA pathway and their regulation.

Enzymes: PhaA, β-ketothiolase; PhaB, acetoacetyl-CoA reductase; CroR, crotonase; Ccr, crotonyl-CoA carboxylase/reductase; Epi, ethylmalonyl-CoA/methylmalonyl-CoA epimerase; Ecm, ethylmalonyl-CoA mutase; Msd, methylsuccinyl-CoA dehydrogenase; Mcd, mesaconyl-CoA hydratase; Mcl1, malyl-CoA/β-methylmalyl-CoA lyase. The y axis is in nmol min⁻¹ mg⁻¹ protein. M, methanol grown cells; A, acetate grown cells; S, succinate grown cells.

Figure 4. Specific activity of enzymes of the C₂ specific steps of the ethylmalonyl-CoA pathway and their regulation.

Enzymes: Pcc, propionyl-CoA carboxylase; Epi, ethylmalonyl-CoA/methylmalonyl-CoA epimerase; Mcm, methylmalonyl-CoA mutase; Mcl2, malyl-CoA thioesterase. The y axis is in nmol min⁻¹ mg⁻¹ protein. M, methanol grown cells; A, acetate grown cells; S, succinate grown cells.

Figure 5. Postulated limiting steps in methanol assimilation in M. extorquens AM1.

The grey dashed lines indicate the calculated minimal value for the specific activity of enzymes, which is required to account for the observed generation time of 3 hours on methanol.