13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-l-methionine production

Hayakawa, Kenshi; Mizutani, Fumio; Shimizu, Hiroshi

doi:10.1186/s12934-018-0935-6

Cited by 14 publications

(8 citation statements)

References 48 publications

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“…2 mg each) were resuspended in 600μl of 6N HCl. The samples were incubated at 100°C for 18 h to hydrolyze the protein and get the amino acid released (42). The hydrolysates were centrifuged and 50μl supernatant was transferred to fresh vial and subjected to vacuum drying in a speed-vacuum (Thermo scientific) to ensure the complete removal of moisture.…”

Section: Acid Hydrolysis Of Cell Pellets and Derivatization Of Amino mentioning

confidence: 99%

Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism

Bhatia

Thakur

Suyal

et al. 2020

Journal of Biological Chemistry

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Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by S-adenosyl methionine (SAM) for decades, but the importance of this regulatory control to one carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. 13C isotope labelling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CH3THF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling, and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one carbon metabolism with various pathways both in yeasts and in humans.

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Section: Acid Hydrolysis Of Cell Pellets and Derivatization Of Amino mentioning

confidence: 99%

Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism

Bhatia

Thakur

Suyal

et al. 2020

Journal of Biological Chemistry

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“…The selection of ethanol or isopropanol did not seem to be of consequence, either, as the observed molar amounts were not consistently higher for one solvent regarding all or even most amino acids. Unlike isopropanol, however, ethanol may be given consideration as a substrate in ILE [ 42 ], in which case the addition of an ethanol solution for quenching would at the very least alter the labeling pattern of the ethanol pool. Thus, the setup with isopropanol was henceforth used as described in more detail in the “ Methods ” section.…”

Section: Resultsmentioning

confidence: 99%

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments

et al. 2022

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Background Currently, the generation of genetic diversity for microbial cell factories outpaces the screening of strain variants with omics-based phenotyping methods. Especially isotopic labeling experiments, which constitute techniques aimed at elucidating cellular phenotypes and supporting rational strain design by growing microorganisms on substrates enriched with heavy isotopes, suffer from comparably low throughput and the high cost of labeled substrates. Results We present a miniaturized, parallelized, and automated approach to 13C-isotopic labeling experiments by establishing and validating a hot isopropanol quenching method on a robotic platform coupled with a microbioreactor cultivation system. This allows for the first time to conduct automated labeling experiments at a microtiter plate scale in up to 48 parallel batches. A further innovation enabled by the automated quenching method is the analysis of free amino acids instead of proteinogenic ones on said microliter scale. Capitalizing on the latter point and as a proof of concept, we present an isotopically instationary labeling experiment in Corynebacterium glutamicum ATCC 13032, generating dynamic labeling data of free amino acids in the process. Conclusions Our results show that a robotic liquid handler is sufficiently fast to generate informative isotopically transient labeling data. Furthermore, the amount of biomass obtained from a sub-milliliter cultivation in a microbioreactor is adequate for the detection of labeling patterns of free amino acids. Combining the innovations presented in this study, isotopically stationary and instationary automated labeling experiments can be conducted, thus fulfilling the prerequisites for 13C-metabolic flux analyses in high-throughput.

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“…Tomàs-Gamisans et al [ 26 ] found that glycerol as a sole carbon source could promote amino acid production by Pichia pastoris, by metabolic flux analysis. Hayakawa et al [ 27 ] conducted a metabolic model of adding ethanol to promote S-adenosyl-L-methionine production in Saccharomyces cerevisiae and found that the metabolic flux levels of the tricarboxylic acid cycle and glyoxylate shunt in the ethanol culture were significantly higher than that in the glucose culture, and more ATP was produced from ethanol by oxidative phosphorylation. However, to our knowledge, there are no reports in the existing literature on this type of research, due to the complexity of the metabolites of edible and medicinal fungi and the lack of in-depth research on metabolic networks.…”

Section: Discussionmentioning

confidence: 99%

Effects of Oleic Acid Addition Methods on the Metabolic Flux Distribution of Ganoderic Acids R, S and T’s Biosynthesis

Yan

Liu

et al. 2022

JoF

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The effects of oleic acid addition methods on the metabolic flux distribution of ganoderic acids R, S and T’s biosynthesis from Ganoderma lucidum were investigated. The results showed that adding filter-sterilized oleic acid in the process of submerged fermentation and static culture is of benefit to the synthesis of ganoderic acids R, S and T. The metabolic fluxes were increased by 97.48%, 78.42% and 43.39%, respectively. The content of ganoderic acids R, S and T were 3.11 times, 5.19 times and 1.44 times higher, respectively, than they were in the control group, which was without additional oleic acid. Ganoderic acids R, S and T’s synthesis pathways (GAP), tricarboxylic acid cycles (TCA), pentose phosphate pathways (PP) and glycolysis pathways (EMP) were all enhanced in the process. Therefore, additional oleic acid can strengthen the overall metabolic flux distribution of G. lucidum in a submerged fermentation-static culture and it can reduce the accumulation of the by-product mycosterol. This study has laid an important foundation for improving the production of triterpenes in the submerged fermentation of G. lucidum.

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13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-l-methionine production

Cited by 14 publications

References 48 publications

Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism

Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments

Effects of Oleic Acid Addition Methods on the Metabolic Flux Distribution of Ganoderic Acids R, S and T’s Biosynthesis

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