Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum

Faijes, Magda; Mars, Astrid E.; Smid, Eddy J.

doi:10.1186/1475-2859-6-27

Cited by 153 publications

(141 citation statements)

References 38 publications

(45 reference statements)

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“…One volume of culture broth was mixed with two volumes of cold 60% methanol plus 70 mM HEPES for quenching the intracellular metabolism, according to the methods described previously (Yamauchi et al, 2014;Faijes et al, 2007). Cells were then collected by centrifugation and washed twice with saline, followed by removal of suspended CaCO 3 by flash centrifugation.…”

Section: Determination Of Nadph/nadp+ Ratiomentioning

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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Section: Determination Of Nadph/nadp+ Ratiomentioning

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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“…From the classical functional genomics/systems biology tools, metabolomics is the most recent technique. Unlike for bacteria and yeast (Bolten and Wittmann 2008;Faijes et al 2007;Schaub et al 2006;Villas-Bôas et al 2005), there are no standard protocols for sampling and extraction of suspended animal cells. A number of different rapid sampling methods are currently used (Sellick et al 2009;Wiendahl et al 2007;Dietmair et al 2010), but have not been compared to other methods in depth.…”

Section: Introductionmentioning

confidence: 99%

Fast filtration for metabolome sampling of suspended animal cells

Volmer

Northoff²,

Scholz

et al. 2010

Biotechnol Lett

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“…Simultaneously, the quenched bacteria cells were extracted by 2 ml boiling ethanol/water (3:1, v/v) solution (95 °C) according to Faijes et al (2007). After that, the preparations were centrifugated (10 000g, 5 min, −20 °C), and the supernatant was frozen at −80 °C and used as an intracellular sample for analyzing the metabolite levels in L. bulgaricus.…”

Section: Quenching Processmentioning

confidence: 99%

Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus

Chen

Sun

et al. 2014

J. Zhejiang Univ. Sci. B

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Abstract:This study proposed a quenching protocol for metabolite analysis of Lactobacillus delbrueckii subsp. bulgaricus. Microbial cells were quenched with 60% methanol/water, 80% methanol/glycerol, or 80% methanol/water. The effect of the quenching process was assessed by the optical density (OD)-based method, flow cytometry, and gas chromatography-mass spectrometry (GC-MS). The principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were employed for metabolite identification. The results indicated that quenching with 80% methanol/water solution led to less damage to the L. bulgaricus cells, characterized by the lower relative fraction of prodium iodide (PI)-labeled cells and the higher OD recovery ratio. Through GC-MS analysis, higher levels of intracellular metabolites (including focal glutamic acid, aspartic acid, alanine, and AMP) and a lower leakage rate were detected in the sample quenched with 80% methanol/water compared with the others. In conclusion, we suggested a higher concentration of cold methanol quenching for L. bulgaricus metabolomics due to its decreasing metabolite leakage.

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Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum

Abstract: Background: A reliable quenching and metabolite extraction method has been developed for Lactobacillus plantarum. The energy charge value was used as a critical indicator for fixation of metabolism.

Cited by 153 publications

References 38 publications

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

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

Fast filtration for metabolome sampling of suspended animal cells

Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus

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