Microbial metabolism of C1 and C2 compounds. The role of glyoxylate, glycollate and acetate in the growth of Pseudomonas AM1 on ethanol and on C1 compounds

Abstract

Succinate (or a product of succinate metabolism) is a catabolite repressor of some enzymes of the serine pathway (hydroxypyruvate reductase, serine-glyoxylate aminotransferase and glycerate kinase) but not of methanol dehydrogenase nor methylamine dehydrogenase. A mutant (PCT64) of Pseudomonas AM1, which is unable to grow on C(1) compounds, lacks glycerate kinase, showing that this enzyme is essential for the operation of the serine pathway. Mutant PCT48, unable to convert acetate into glycollate, has lost the ability to grow both on C(1) compounds and on ethanol. The properties of a third mutant (PCT57) show that Pseudomonas AM1 contains enzymes catalysing the conversion of acetate into glyoxylate. Evidence is presented that hydroxypyruvate reductase is involved in the oxidation of glycollate to glyoxylate during growth on ethanol. A scheme is proposed for the conversion of ethanol and of C(1) compounds into glyoxylate in which acetate (or a derivative) and glycollate are intermediates.

Demonstration of the ethylmalonyl-CoA pathway by using 13 C metabolomics

“…After 10 s of incubation, the percentage of 13 C label incorporated into ethylmalonyl-CoA did not increase further, unlike other CoA metabolites (Fig. 4).…”

“…Instead of the glyoxylate cycle, this process is achieved by a not yet established pathway where CoA thioesters are known to play a key role. We applied state-of-the-art metabolomics and 13 C metabolomics strategies to demonstrate how glyoxylate is generated during methylotrophic growth in the isocitrate lyase-negative methylotroph Methylobacterium extorquens AM1. High-resolution mass spectrometry showed the presence of CoA thioesters specific to the recently proposed ethylmalonyl-CoA pathway.…”

“…The assimilation of C1 units by this pathway requires continuous regeneration of glyoxylate from acetyl-CoA and can be achieved, in principle, via the well-known glyoxylate cycle (10). However, Dunstan and coworkers (11)(12)(13)(14) showed in 1972 and 1973 that M. extorquens AM1 lacks the key enzyme of the glyoxylate cycle, isocitrate lyase, but has an alternative route involving oxidation of acetate to glyoxylate that functions during growth on both C1 and C2 compounds. Also, other organisms, including the photosynthetic Rhodobacter sphaeroides are known to require an alternative to the glyoxylate cycle when growing on C2 substrates or on substrates that are converted into acetyl-CoA to enter central metabolism (15-18).…”

“…The development of an original liquid chromatography high-resolution mass spectrometry (LC-HRMS) method allowed for the identification of almost all intermediates of the ethylmalonyl-CoA pathway. Detailed and conclusive information regarding the operation of the ethylmalonyl-CoA pathway as the predominant process for glyoxylate formation was obtained from 13 C-labeling experiments in which kinetic isotopomer profiles collected by LC-HRMS during short-term 13 C-labeling experiments were combined with steady-state isotopomer distributions measured by NMR.…”

Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1

“…Succinate (20 mM), methanol (100 mM), methylamine (20 mM), ethylamine (20 mM), formate (40 mM), pyruvate (40 mM) or P-hydroxybutyrate (20 mM) were used as substrates. Methanol induction of mutants was carried out as described by Dunstan et al (1972b). The following antibiotic concentrations were used for M. extorquens AM1 : tetracycline (Tc), 10 pg ml-'; kanamycin (Km), 100 pg ml-'; and rifamycin, 50 pg ml-'.…”