<sup>13</sup>C NMR Studies of Histidine Fermentation with a<i>Corynebacterium glutamicum</i>Mutant

Ishino, Shuichi; Kuga, Tetsurō; Yamaguchi, Kazuo; Shirahata, Kunikatsu; Araki, Kazumi

doi:10.1080/00021369.1986.10867392

Cited by 3 publications

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References 2 publications

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“…5). Furthermore, strains C. glutamicum HIS1–HIS8 secreted glycine as inevitable equimolar byproduct to histidine, which has also been observed for other histidine producing mutants of C. glutamicum and Brevibacterium flavum [19, 44].…”

Section: Discussionmentioning

confidence: 59%

Modular systems metabolic engineering enables balancing of relevant pathways for l-histidine production with Corynebacterium glutamicum

Schwentner

Feith

Münch

et al. 2019

Biotechnol Biofuels

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Backgroundl-Histidine biosynthesis is embedded in an intertwined metabolic network which renders microbial overproduction of this amino acid challenging. This is reflected in the few available examples of histidine producers in literature. Since knowledge about the metabolic interplay is limited, we systematically perturbed the metabolism of Corynebacterium glutamicum to gain a holistic understanding in the metabolic limitations for l-histidine production. We, therefore, constructed C. glutamicum strains in a modularized metabolic engineering approach and analyzed them with LC/MS-QToF-based systems metabolic profiling (SMP) supported by flux balance analysis (FBA).ResultsThe engineered strains produced l-histidine, equimolar amounts of glycine, and possessed heavily decreased intracellular adenylate concentrations, despite a stable adenylate energy charge. FBA identified regeneration of ATP from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as crucial step for l-histidine production and SMP identified strong intracellular accumulation of inosine monophosphate (IMP) in the engineered strains. Energy engineering readjusted the intracellular IMP and ATP levels to wild-type niveau and reinforced the intrinsic low ATP regeneration capacity to maintain a balanced energy state of the cell. SMP further indicated limitations in the C1 supply which was overcome by expression of the glycine cleavage system from C. jeikeium. Finally, we rerouted the carbon flux towards the oxidative pentose phosphate pathway thereby further increasing product yield to 0.093 ± 0.003 mol l-histidine per mol glucose.ConclusionBy applying the modularized metabolic engineering approach combined with SMP and FBA, we identified an intrinsically low ATP regeneration capacity, which prevents to maintain a balanced energy state of the cell in an l-histidine overproduction scenario and an insufficient supply of C1 units. To overcome these limitations, we provide a metabolic engineering strategy which constitutes a general approach to improve the production of ATP and/or C1 intensive products.

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Section: Discussionmentioning

confidence: 59%

Modular systems metabolic engineering enables balancing of relevant pathways for l-histidine production with Corynebacterium glutamicum

Schwentner

Feith

Münch

et al. 2019

Biotechnol Biofuels

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Pathways at Work: Metabolic Flux Analysis of the Industrial Cell Factory Corynebacterium glutamicum

Becker¹,

Wittmann²

2020

Corynebacterium Glutamicum

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Streamlining the Analysis of Dynamic 13C-Labeling Patterns for the Metabolic Engineering of Corynebacterium glutamicum as l-Histidine Production Host

et al. 2020

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Today’s possibilities of genome editing easily create plentitudes of strain mutants that need to be experimentally qualified for configuring the next steps of strain engineering. The application of design-build-test-learn cycles requires the identification of distinct metabolic engineering targets as design inputs for subsequent optimization rounds. Here, we present the pool influx kinetics (PIK) approach that identifies promising metabolic engineering targets by pairwise comparison of up- and downstream 13C labeling dynamics with respect to a metabolite of interest. Showcasing the complex l-histidine production with engineered Corynebacterium glutamicuml-histidine-on-glucose yields could be improved to 8.6 ± 0.1 mol% by PIK analysis, starting from a base strain. Amplification of purA, purB, purH, and formyl recycling was identified as key targets only analyzing the signal transduction kinetics mirrored in the PIK values.

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¹³C NMR Studies of Histidine Fermentation with aCorynebacterium glutamicumMutant

Cited by 3 publications

References 2 publications

Modular systems metabolic engineering enables balancing of relevant pathways for l-histidine production with Corynebacterium glutamicum

Modular systems metabolic engineering enables balancing of relevant pathways for l-histidine production with Corynebacterium glutamicum

Pathways at Work: Metabolic Flux Analysis of the Industrial Cell Factory Corynebacterium glutamicum

Streamlining the Analysis of Dynamic 13C-Labeling Patterns for the Metabolic Engineering of Corynebacterium glutamicum as l-Histidine Production Host

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13C NMR Studies of Histidine Fermentation with aCorynebacterium glutamicumMutant

Cited by 3 publications

References 2 publications

Modular systems metabolic engineering enables balancing of relevant pathways for l-histidine production with Corynebacterium glutamicum

Modular systems metabolic engineering enables balancing of relevant pathways for l-histidine production with Corynebacterium glutamicum

Pathways at Work: Metabolic Flux Analysis of the Industrial Cell Factory Corynebacterium glutamicum

Streamlining the Analysis of Dynamic 13C-Labeling Patterns for the Metabolic Engineering of Corynebacterium glutamicum as l-Histidine Production Host

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¹³C NMR Studies of Histidine Fermentation with aCorynebacterium glutamicumMutant