Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate

Witthoff, Sabrina; Eggeling, Lothar; Bott, Michael; Polen, Tino

doi:10.1099/mic.0.059196-0

Cited by 20 publications

(15 citation statements)

References 87 publications

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“…The quantitative measurement of methanol and glucose in the cell-free supernatant of C. glutamicum cultures was performed by capillary gas chromatography (GC) using an Agilent 7890A gas chromatograph (Agilent Technologies, Waldbronn, Germany) and by high-performance liquid chromatography (HPLC) analysis using an Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, Germany), respectively, as described previously (22,39). Enzyme assays of crude extracts.…”

Section: Methodsmentioning

confidence: 99%

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Witthoff

Schmitz

Niedenführ

et al. 2015

Appl Environ Microbiol

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Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ؎ 0.3 mM/h (mean ؎ standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13 C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m ؉ 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum ⌬ald ⌬adhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO 2 , the m ؉ 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

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Section: Methodsmentioning

confidence: 99%

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Witthoff

Schmitz

Niedenführ

et al. 2015

Appl Environ Microbiol

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“…Formaldehyde can be oxidized to formate either by acetaldehyde dehydrogenase (Ald) or by mycothioldependent formaldehyde dehydrogenase (FadH) Witthoff et al 2013). Subsequently, formate is oxidized to CO 2 by formate dehydrogenase FdhF (cg0618), involving a currently unknown electron acceptor and the gene products encoded by cg0616 and cg0617 (Witthoff et al 2012). Recently, assimilation of methanol into central metabolites by engineered bacteria has been described for Pseudomonas putida (Koopman et al 2009), Escherichia coli (Müller et al 2015b), and also C. glutamicum (Witthoff et al 2015).…”

Section: Introductionmentioning

confidence: 98%

Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate

Leßmeier

Pfeifenschneider

Carnicer

et al. 2015

Appl Microbiol Biotechnol

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Methanol, a one-carbon compound, can be utilized by a variety of bacteria and other organisms as carbon and energy source and is regarded as a promising substrate for biotechnological production. In this study, a strain of non-methylotrophic Corynebacterium glutamicum, which was able to produce the polyamide building block cadaverine as non-native product, was engineered for co-utilization of methanol. Expression of the gene encoding NAD+-dependent methanol dehydrogenase (Mdh) from the natural methylotroph Bacillus methanolicus increased methanol oxidation. Deletion of the endogenous aldehyde dehydrogenase genes ald and fadH prevented methanol oxidation to carbon dioxide and formaldehyde detoxification via the linear formaldehyde dissimilation pathway. Heterologous expression of genes for the key enzymes hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase of the ribulose monophosphate (RuMP) pathway in this strain restored growth in the presence of methanol or formaldehyde, which suggested efficient formaldehyde detoxification involving RuMP key enzymes. While growth with methanol as sole carbon source was not observed, the fate of 13C-methanol added as co-substrate to sugars was followed and the isotopologue distribution indicated incorporation into central metabolites and in vivo activity of the RuMP pathway. In addition, 13C-label from methanol was traced to the secreted product cadaverine. Thus, this synthetic biology approach led to a C. glutamicum strain that converted the non-natural carbon substrate methanol at least partially to the non-native product cadaverine.

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“…On the other hand, the genome encodes all enzymes for THF-dependent formaldehyde oxidation, involving genes encoding a putative bifunctional methylene-THF dehydrogenase/cyclohydrolase (cg0750) and a putative formyl-THF deformylase (cg0457). Recently, a formate dehydrogenase has been discovered in C. glutamicum (Witthoff et al, 2012). The electron acceptor of the enzyme encoded in the fdhFcg0617-fdhD cluster remains unknown.…”

Section: Introductionmentioning

confidence: 99%

“…The electron acceptor of the enzyme encoded in the fdhFcg0617-fdhD cluster remains unknown. The molybdenumcontaining enzyme FdhF oxidizes formate to carbon dioxide and reduces growth retardation caused by formate, which can be found in many environments (Witthoff et al, 2012). Since factor-independent and thiol-dependent formaldehyde detoxification yields formate which can be oxidized to carbon dioxide by FdhF in C. glutamicum, we tested formaldehyde toxicity and formaldehyde oxidation in C. glutamicum and sought to identify and characterize the involved enzyme(s).…”

Section: Introductionmentioning

confidence: 99%

Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase

2013

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Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the actinomycetes, is able to degrade formaldehyde but the enzyme(s) involved in this detoxification process were not known. Acetaldehyde dehydrogenase Ald, which is essential for ethanol utilization, and FadH, characterized here as NAD-linked mycothiol-dependent formaldehyde dehydrogenase, were shown to be responsible for formaldehyde oxidation since a mutant lacking ald and fadH could not oxidize formaldehyde resulting in the inability to grow when formaldehyde was added to the medium. Moreover, C. glutamicum DaldDfadH did not grow with vanillate, a carbon source giving rise to intracellular formaldehyde. FadH from C. glutamicum was purified from recombinant Escherichia coli and shown to be active as a homotetramer. Mycothiol-dependent formaldehyde oxidation revealed K m values of 0.6 mM for mycothiol and 4.3 mM for formaldehyde and a V max of 7.7 U mg "1. FadH from C. glutamicum also possesses zinc-dependent, but mycothiolindependent alcohol dehydrogenase activity with a preference for short chain primary alcohols such as ethanol (K m 5330 mM, V max 59.6 U mg "1 ), 1-propanol (K m 5150 mM, V max 55 U mg "1 ) and 1-butanol (K m 550 mM, V max 50.8 U mg "1). Formaldehyde detoxification system by Ald and mycothiol-dependent FadH is essential for tolerance of C. glutamicum to external stress by free formaldehyde in its habitat and for growth with natural substrates like vanillate, which are metabolized with concomitant release of formaldehyde.

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Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate

Cited by 20 publications

References 87 publications

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate

Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase

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