Changes of Pentose Phosphate Pathway Flux in Vivo in Corynebacterium glutamicum during Leucine-Limited Batch Cultivation as Determined from Intracellular Metabolite Concentration Measurements

Moritz, Bernd; Striegel, Katharina; Graaf, Albert A. de; Sahm, Hermann

doi:10.1006/mben.2002.0233

Cited by 33 publications

(22 citation statements)

References 31 publications

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“…[13][14][15] On the other hand, the feature observed for the amino acid-biosynthetic pathways was unexpected, because those up-regulated genes included genes such as lysC-asd, serAB, and aroEBKC, for which no transcriptional regulation is known. It is interesting to note that the expression levels varied not more than 10-fold in most of those genes.…”

Section: Communicationmentioning

confidence: 99%

Transcriptome Analysis Reveals Global Expression Changes in an IndustrialL-Lysine Producer ofCorynebacterium glutamicum

Hayashi¹,

Ohnishi²,

Mitsuhashi³

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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Section: Communicationmentioning

confidence: 99%

Transcriptome Analysis Reveals Global Expression Changes in an IndustrialL-Lysine Producer ofCorynebacterium glutamicum

Hayashi¹,

Ohnishi²,

Mitsuhashi³

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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“…glutamicum (Moritz et al, 2002). It has been reported in this organism that NADPH is generated mainly through the oxidative pentose phosphate pathway (PPP) (Marx et al, 1996).…”

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confidence: 99%

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Takeno

Hori

Ohtani

et al. 2016

Metabolic Engineering

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We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for L-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of L-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the 2 myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A iol was engineered into an L-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of L-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect L-lysine production in the engineered GapN strains, while a drastic decrease in L-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP + ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient L-lysine production independent of the oxidative pentose phosphate pathway.

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“…For efficient production of L-lysine, the supply of cofactor NADPH is one of the critical factors, because 1 mol of L-lysine requires 4 mol of NADPH for its biosynthesis from glucose in C. glutamicum (23). For this organism, it has been reported that NADPH is generated mainly through an oxidative pentose phosphate pathway (PPP) and partly by NADP-dependent isocitrate dehydrogenase and NADP-dependent malic enzyme (17,18,19).…”

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confidence: 99%

Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l -Lysine Production

Takeno

Murata

Kobayashi

et al. 2010

Appl Environ Microbiol

105

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A sufficient supply of NADPH is a critical factor in L-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP ؉ to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an L-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an L-lysine producer was evaluated. The amounts of L-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ϳ70% on glucose, ϳ120% on fructose, and ϳ100% on sucrose, indicating that the increased L-lysine production was attributed to GapN. These results demonstrate effective L-lysine production by C. glutamicum with an additional source of NADPH during glycolysis.

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Changes of Pentose Phosphate Pathway Flux in Vivo in Corynebacterium glutamicum during Leucine-Limited Batch Cultivation as Determined from Intracellular Metabolite Concentration Measurements

Cited by 33 publications

References 31 publications

Transcriptome Analysis Reveals Global Expression Changes in an IndustrialL-Lysine Producer ofCorynebacterium glutamicum

Transcriptome Analysis Reveals Global Expression Changes in an IndustrialL-Lysine Producer ofCorynebacterium glutamicum

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l -Lysine Production

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