The prediction that malate synthase activity is required for M. extorquens
AM1 to grow on C2
compounds via the EMC pathway stands in contrast to the literature evidence that a mutant lacking malate synthase activity still is able to grow on C2
). Possible explanations for this contradiction are the following: (i) the EMC pathway does not operate during growth on C2
compounds, (ii) glyoxylate or a product made from glyoxylate is excreted, (iii) malate synthase activity actually is present in the malyl-CoA lyase mutant grown on C2
compounds, or (iv) an alternative glyoxylate consumption pathway exists.
Explanation i clearly is not correct, since a large body of evidence has shown that M. extorquens
AM1 grows on C2
compounds using the same pathway that is involved in converting acetyl-CoA to glyoxylate during growth on C1
). Most compelling is the fact that mutants in this pathway are defective for growth on both C1
compounds and are rescued for growth on both C1
compounds by the addition of glyoxylate (11
). The results presented here are consistent with the operation of the EMC pathway during growth on ethylamine. Most of the genes involved in the conversion of acetyl-CoA to glyoxylate and propionyl-CoA by the EMC pathway showed higher expression in cells grown on ethylamine than in cells grown on succinate, similarly to the result found with methanol-grown cells (42
) (Fig. , Table ).
Likewise, the utilization of propionyl-CoA during growth on C2
compounds has been suggested to follow a standard conversion scheme via propionyl-CoA carboxylase and methylmalonyl-CoA mutase, succinate dehydrogenase, and fumarase (Fig. ) (30
), and mutants in these genes also show defects in growth on both C1
compounds that are rescued by the addition of glyoxylate or glycolate (7
). Of these genes, only those involved in generating active methylmalonyl-CoA mutase showed higher expression in cells grown on ethylamine than succinate. However, the slow growth observed on C2
compounds does not require high enzyme levels to support the flux required. It can be calculated that the flux through this pathway required to support a 12-h doubling time is approximately 15 nmol min−1
, and these enzymes are present in succinate-grown cells at in vitro
activities greater than this (11
). These results all are consistent with the previous mutant data showing that the EMC pathway must operate for the growth of M. extorquens
AM1 on C2
For explanation ii, the excretion of glyoxylate or related compounds would drop the yield, but it could allow the cells to grow on the propionyl-CoA generated from ß-methylmalyl-CoA. About 40% of the total carbon flux to biomass goes to glyoxylate via the EMC pathway, so the amount to be excreted would be significant. M. extorquens
AM1 is known to have in vitro
activities for interconverting glyoxylate and glycolate (3
) and interconverting glyoxylate and oxalyl-CoA, which then can be converted to oxalate (5
), although not all of the genes involved in these activities have been identified. Measurements of intracellular and extracellular glyoxylate, glycolate, and oxalate showed that these compounds all were detectable in the wild type both intracellularly and extracellularly. However, the mclA1
mutant did not show major differences in the levels of these compounds compared to those of the wild type, either intracellularly or extracellularly, suggesting that the excretion or accumulation of glyoxylate or products derived from glyoxylate does not explain the growth of the mclA1
mutant on C2
compounds. It is notable that the intracellular levels of glyoxylate in the mclA1
mutant were not significantly different from those of the wild type, suggesting that an alternative glyoxylate consumption route exists. Although the metabolite detection methods used here did not determine absolute concentrations, based on previous analyses (52
) it can be estimated that the total amount of glyoxylate, glycolate, and oxalate detected in the supernatant of the mclA1
mutant was at least two orders of magnitude lower than expected if the glyoxylate flux was directed to excretion.
Explanation iii, that the mclA1 mutant contains malate synthase activity during growth on C2 compounds, is at best only a partial explanation as, based on Mcl activity measurements, the low activity would not account for the utilization of all of the glyoxylate generated via the EMC pathway and would not support the 25-h doubling time of this mutant on ethylamine (approximately 7.5 nmol min−1 mg protein−1). However, we did show that the growth of the mclA1 deletion mutant is only partially impaired on ethylamine, suggesting that MclA2 performs a similar function but at a reduced level. The existence of this isoenzyme thus provides a partial solution to the conundrum.
Explanation iv, the presence of an alternative glyoxylate consumption pathway, was shown to provide the final solution to the conundrum. This pathway involves sga, glyA, and gcv, and it appears to operate not only in mutants defective in the EMC pathway but also in the wild type during growth on C2 compounds. Evidence for this pathway was obtained by observing mutant phenotypes and was consistent with labeling experiments involving both mutants and the wild type, as described above. Further confirmation of the importance of this glycine/serine pathway during growth on C2 compounds was obtained from the finding that both mclA1 sga and mclA2 sga double mutants have (partially) impaired glyoxylate consumption routes and show significant growth defects on ethylamine, unlike each of the single mutants.
Once glyoxylate is converted to serine, it has multiple routes for incorporation, including conversion to protein and to other C2
, and C4
compounds via the routes shown in Fig. . Strains containing mutations in some steps of these pathways are known to grow on C2
compounds, including strains lacking hpr
) and gck
), suggesting either that serine is routed into central metabolites via alternative pathways or that, in vivo
, other enzymes are present that carry out this function at the low flux required to support the observed growth rate. A possible pathway involving these steps can be drawn for converting 2-glyoxylate to a malate (Fig. and ), using known enzymes and genes in M. extorquens
AM1, although it is energetically expensive, requiring one NADH molecule and one ATP molecule per malate molecule, and even more if an energy-requiring transamination reaction is involved. However, glyoxylate is a reactive aldehyde and is inhibitory to M. extorquens
AM1 above 2 mM in the external medium. Accumulation inside the cell is likely to be detrimental, and it is possible that the slow growth observed on C2
compounds in the wild type is due in part to the tradeoff between the detriments of glyoxylate accumulation and the low yield involved in this pathway. Our results show that even in the mutants analyzed here, glyoxylate does not accumulate, pointing to a careful control system to keep it from building up.
In summary, our results demonstrate that during growth on ethylamine, M. extorquens AM1 uses an alternative route for glyoxylate consumption via glycine and serine to complement the expected malate synthase route. Neither pathway alone supports wild-type growth, but the combination allows this bacterium to grow normally on C2 compounds. This finding suggests that the two-step malate synthase reaction in M. extorquens AM1 creates a bottleneck for glyoxylate consumption, which the cell has overcome by shunting glyoxylate through a second pathway. Although the measured in vitro activity of malate synthase (16 nmol min−1 mg protein−1 for ethylamine-grown cells) should be just sufficient to support the growth rate on C2 compounds, our results show that the in vivo activities must restrict flux through this route. The presence of alternative routes for the consumption of a toxic intermediate is a logical metabolic strategy and demonstrates the versatility and flexibility of the metabolic network in this facultative methylotroph. In addition, it represents a possible model for other metabolic networks involving high flux through a toxic intermediate.