OdhI dephosphorylation kinetics during different glutamate production processes involving Corynebacterium glutamicum

ABSTRACTMethanol 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 bacteriumCorynebacterium glutamicumtoward 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 fromBacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway ofBacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinantC. glutamicumstrain 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, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of aC. glutamicumΔaldΔadhEmutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineeredC. glutamicumstrains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

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Metabolic Analysis of Glutamate Production by Corynebacterium glutamicum

Metabolic Engineering ◽

10.1006/mben.1999.0122 ◽

1999 ◽

Vol 1 (3) ◽

pp. 224-231 ◽

Cited By ~ 33

Author(s):

Pierre Gourdon ◽

Nicholas D. Lindley

Keyword(s):

Corynebacterium Glutamicum ◽

Metabolic Analysis ◽

Glutamate Production

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Enhancing the supply of oxaloacetate for l-glutamate production by pyc overexpression in different Corynebacterium glutamicum

Biotechnology Letters ◽

10.1007/s10529-013-1241-3 ◽

2013 ◽

Vol 35 (6) ◽

pp. 943-950 ◽

Cited By ~ 7

Author(s):

Xuan Guo ◽

Jing Wang ◽

Xixian Xie ◽

Qingyang Xu ◽

Chenglin Zhang ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Glutamate Production

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Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.5920-5928.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 5920-5928 ◽

Cited By ~ 132

Author(s):

Corinna Stansen ◽

Davin Uy ◽

Stephane Delaunay ◽

Lothar Eggeling ◽

Jean-Louis Goergen ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Carbon Source ◽

Corynebacterium Glutamicum ◽

Specific Activity ◽

Temperature Shift ◽

Pcr Analysis ◽

Mrna Levels ◽

Reverse Transcription Pcr ◽

Glutamate Production ◽

Lactate Utilization

ABSTRACT Gene expression changes of glutamate-producing Corynebacterium glutamicum were identified in transcriptome comparisons by DNA microarray analysis. During glutamate production induced by a temperature shift, C. glutamicum strain 2262 showed significantly higher mRNA levels of the NCgl2816 and NCgl2817 genes than its non-glutamate-producing derivative 2262NP. Reverse transcription-PCR analysis showed that the two genes together constitute an operon. NCgl2816 putatively codes for a lactate permease, while NCgl2817 was demonstrated to encode quinone-dependent l-lactate dehydrogenase, which was named LldD. C. glutamicum LldD displayed Michaelis-Menten kinetics for the substrate l-lactate with a Km of about 0.51 mM. The specific activity of LldD was about 10-fold higher during growth on l-lactate or on an l-lactate-glucose mixture than during growth on glucose, d-lactate, or pyruvate, while the specific activity of quinone-dependent d-lactate dehydrogenase differed little with the carbon source. RNA levels of NCgl2816 and lldD were about 18-fold higher during growth on l-lactate than on pyruvate. Disruption of the NCgl2816-lldD operon resulted in loss of the ability to utilize l-lactate as the sole carbon source. Expression of lldD restored l-lactate utilization, indicating that the function of the permease gene NCgl2816 is dispensable, while LldD is essential, for growth of C. glutamicum on l-lactate.

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