Engineering of Corynebacterium glutamicum towards utilization of methanol as carbon and energy source
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Methanol is a pure and inexpensive raw material, which is mainly produced from fossil-fuelbased synthesis gas. Over the past years, new approaches were developed for its production from renewable carbon sources. In the chemical industry, methanol is already an important carbon feedstock, but it has found only limited application in biotechnology. This can predominantly be attributed to the inability of important microbial platform organisms to utilize this C1 compound. With the aim to make methanol a suitable substrate for microbial production processes, the non-methylotrophic and industrially important amino acid-producing bacterium Corynebacterium glutamicum was engineered towards the utilization of methanol as auxiliary carbon source in a sugar-based medium. Initial experiments on the response of C. glutamicum to methanol showed that this organism is able to oxidize methanol to CO2 during the stationary phase with a rate of 0.83 ± 0.2 mM/h (2.8 ± 0.5 nmol min-1 mg CDW-1) in glucose/methanol defined medium. Methanol oxidation was shown to be subject to carbon catabolite repression in the presence of glucose and to be dependent on the transcriptional regulator RamA. Global gene expression studies revealed that the alcohol dehydrogenase gene adhA as well as the aldehyde dehydrogenase gene ald were upregulated in the presence of methanol. Analysis of a mutant lacking the adhA gene showed a 67% reduced methanol consumption rate (0.27 ± 0.05 mM/h), indicating that AdhA is mainly responsible for the oxidation of methanol to formaldehyde. The oxidation of formaldehyde to formate was found to be catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. A double mutant lacking ald and adhE was severely impaired in its ability to oxidize formaldehyde. The oxidation of formate to CO2 is catalyzed by formate dehydrogenase (FDH). Deletion of fdhF (annotated as FDH) and fdhD (annotated as FDH accessory protein) in C. glutamicum abolished formate oxidation and resulted in an increased formate sensitivity. Growth studies with molybdenum and tungsten indicated that FdhF is a molybdenumdependent enzyme. The electron acceptor of FdhF is not NAD(P)+ and still unknown
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Engineering of Corynebacterium glutamicum towards utilization of methanol as carbon and energy source, Sabrina Witthoff
- Sprache
- Erscheinungsdatum
- 2015
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- Titel
- Engineering of Corynebacterium glutamicum towards utilization of methanol as carbon and energy source
- Sprache
- Englisch
- Autor*innen
- Sabrina Witthoff
- Erscheinungsdatum
- 2015
- ISBN10
- 3958060293
- ISBN13
- 9783958060296
- Kategorie
- Skripten & Universitätslehrbücher
- Beschreibung
- Methanol is a pure and inexpensive raw material, which is mainly produced from fossil-fuelbased synthesis gas. Over the past years, new approaches were developed for its production from renewable carbon sources. In the chemical industry, methanol is already an important carbon feedstock, but it has found only limited application in biotechnology. This can predominantly be attributed to the inability of important microbial platform organisms to utilize this C1 compound. With the aim to make methanol a suitable substrate for microbial production processes, the non-methylotrophic and industrially important amino acid-producing bacterium Corynebacterium glutamicum was engineered towards the utilization of methanol as auxiliary carbon source in a sugar-based medium. Initial experiments on the response of C. glutamicum to methanol showed that this organism is able to oxidize methanol to CO2 during the stationary phase with a rate of 0.83 ± 0.2 mM/h (2.8 ± 0.5 nmol min-1 mg CDW-1) in glucose/methanol defined medium. Methanol oxidation was shown to be subject to carbon catabolite repression in the presence of glucose and to be dependent on the transcriptional regulator RamA. Global gene expression studies revealed that the alcohol dehydrogenase gene adhA as well as the aldehyde dehydrogenase gene ald were upregulated in the presence of methanol. Analysis of a mutant lacking the adhA gene showed a 67% reduced methanol consumption rate (0.27 ± 0.05 mM/h), indicating that AdhA is mainly responsible for the oxidation of methanol to formaldehyde. The oxidation of formaldehyde to formate was found to be catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. A double mutant lacking ald and adhE was severely impaired in its ability to oxidize formaldehyde. The oxidation of formate to CO2 is catalyzed by formate dehydrogenase (FDH). Deletion of fdhF (annotated as FDH) and fdhD (annotated as FDH accessory protein) in C. glutamicum abolished formate oxidation and resulted in an increased formate sensitivity. Growth studies with molybdenum and tungsten indicated that FdhF is a molybdenumdependent enzyme. The electron acceptor of FdhF is not NAD(P)+ and still unknown