Growth dynamics of a methylotroph (<i>Methylomonas</i> L3) in continuous cultures. I. Fast transients induced by methanol pulses and methanol accumulation

Chu, I‐Ming; Papoutsakis, Eleftherios T.

doi:10.1002/bit.260290109

Cited by 13 publications

(3 citation statements)

References 23 publications

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“…However, while high activities for the enzymes of the H 4 MPT pathway have been measured, mutants in these enzymes revealed only very subtle phenotypes, suggesting a minor role in formaldehyde oxidation. Formate dehydrogenase activity was reported only in cells of M. flagellatus (and other RuMP methylotrophs) when grown in a chemostat at low dilution rates (6,12,13), consistent with the nonessential role of the linear pathway. However, here we detected all the components of the linear pathway, including the two alternative formate dehydrogenases.…”

Section: Resultsmentioning

confidence: 73%

Expressed Genome of Methylobacillus flagellatus as Defined through Comprehensive Proteomics and New Insights into Methylotrophy

et al. 2010

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In recent years, techniques have been developed and perfected for high-throughput identification of proteins and their accurate partial sequencing by shotgun nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS), making it feasible to assess global protein expression profiles in organisms with sequenced genomes. We implemented comprehensive proteomics to assess the expressed portion of the genome of Methylobacillus flagellatus during methylotrophic growth. We detected a total of 1,671 proteins (64% of the inferred proteome), including all the predicted essential proteins. Nonrandom patterns observed with the nondetectable proteins appeared to correspond to silent genomic islands, as inferred through functional profiling and genome localization. The protein contents in methylamine-and methanol-grown cells showed a significant overlap, confirming the commonality of methylotrophic metabolism downstream of the primary oxidation reactions. The new insights into methylotrophy include detection of proteins for the N-methylglutamate methylamine oxidation pathway that appears to be auxiliary and detection of two alternative enzymes for both the 6-phosphogluconate dehydrogenase reaction (GndA and GndB) and the formate dehydrogenase reaction (FDH1 and FDH4). Mutant analysis revealed that GndA and FDH4 are crucial for the organism's fitness, while GndB and FDH1 are auxiliary.

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Section: Resultsmentioning

confidence: 73%

Expressed Genome of Methylobacillus flagellatus as Defined through Comprehensive Proteomics and New Insights into Methylotrophy

et al. 2010

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“…The difficulties which occur when attempting to produce a uniform distribution of methanol throughout a large volume of medium, e.g. a plant fermentor, and the sensitivity of methylotrophs to changes in methanol concentration have been described (Chu and Papoutsakis 1987a;Giuseppin 1988). However, little is known about the physiological responses of methylotrophs to sudden changes in the methanol concentration of the culture media.…”

Section: Introductionmentioning

confidence: 99%

Physiological responses of a methylotrophic bacterium after sudden shifts from C‐limited chemostat to C‐excess batch growth conditions

Gı́rio

Amaral-Collaço

Attwood³

1995

Journal of Applied Bacteriology

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The response of steady‐state continuous cultures of Methylobacterium sp. RXM to the addition of methanol pulses was studied. The increase of methanol concentration in the medium did not result in cell death under any of the conditions tested. When the growth rate of the steady‐state cultures was low (D= 0.046 h−1), the specific growth rate increased. When the concentration of methanol in the pulse was increased from 36 mmol l−1 to 280 mmol l−1, uncoupled growth occurred and the molar cell yield decreased. Conversely, steady‐state cultures at high growth rate (D= 0.2 h−1) showed a decrease in both specific growth rate and molar cell yield after the addition of the methanol pulses (32 and 164 mmol 1−1). For all conditions, formaldehyde and formate were excreted into the medium but the levels did not exceed 1.13 mmol 1−1 Slow‐growing cultures were characterized by cells with high derepressed specific activities of methanol dehydrogenase and low specific activities of formaldehyde and formate dehydrogenases, fast‐growing cells had lower specific activity for methanol dehydrogenase and higher activities of formaldehyde and formate dehydrogenases, resulting in the excretion of lower concentrations of formaldehyde and formate. It is concluded that slow‐growing cultures are more stable than fast‐growing cultures for low methanol concentration fluctuations, and it is expected that maximum growth yields throughout the fermentation time are better achieved under the former conditions. However, for large fluctuations in the substrate concentration, the bacterial metabolic responses were identical both for slow‐growing and fast‐growing cultures.

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“…Since the transport of MeOH is energy dependently2 it is conceivable that when the cells accumulate MeOH to the very high intracellular concentrations estimated in ref. 1, they may be depleting themselves of biosynthetic energy. There is also the possibility that high MeOH concentrations may interfere with the electron transport system, which participates in the biosynthetic energy generation.…”

Section: Introductionmentioning

confidence: 99%

Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. II. Growth inhibition and comparison against an unstructured model

Chu

Papoutsakis

1987

Biotech & Bioengineering

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High methanol concentrations have a negative effect on the growth rate and the biomass yield of growth transients induced by methanol pulses in continuous cultures of Methylomonas L3. The physiological basis of this effect is investigated by measuring the effect of the methanol pulse on the cell energy charge (EC) and ATP, ADP, and AMP concentrations, and by comparing the results of the pulse transients against an unstructured model. The methanol pulse is shown to lead to increased values of the cell EC and ATP concentration, and thus, inhibition and reduced availability of biosynthetic energy are excluded as causes of inhibition. When the biomass and methanol profiles of the transient experiments are compared in phase-plane diagrams against computer simulations based on the model, satisfactory agreement between experimental data and model predictions is found in single-substrate, high-dilution-rate experiments. Conversely, poor agreement between experimental data and simulation results indicates a more severe growth inhibition than the model predicts at low dilution rates and a less severe one in mixed-substrate experiments. Based on these findings and other relevant physiological information, we propose that the large variations in the negative effect of methanol on growth result from the fact that cells accumulate methanol to widely different concentrations depending on their physiological state. In their effort to detoxify from the high intracellular methanol and formaldehyde concentrations, cells oxidize considerably more methanol than they can incorporate into biomass. This leads to a useless ATP surplus, which the cells must hydrolyze without doing any useful biosynthetic work, and this results in lower biomass yields.

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Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. I. Fast transients induced by methanol pulses and methanol accumulation

Cited by 13 publications

References 23 publications

Expressed Genome of Methylobacillus flagellatus as Defined through Comprehensive Proteomics and New Insights into Methylotrophy

Expressed Genome of Methylobacillus flagellatus as Defined through Comprehensive Proteomics and New Insights into Methylotrophy

Physiological responses of a methylotrophic bacterium after sudden shifts from C‐limited chemostat to C‐excess batch growth conditions

Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. II. Growth inhibition and comparison against an unstructured model

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