Metabolome analysis reveals the effect of carbon catabolite control on the poly(γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945

Mitsunaga, Hitoshi; Meissner, Lena; Palmen, Thomas G.; Bamba, Takeshi; Büchs, Jochen; Fukusaki, Eiichiro

doi:10.1016/j.jbiosc.2015.08.012

Cited by 36 publications

(36 citation statements)

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“…From 20 h onwards, the molecular weight decreased steadily throughout the cultivation to a final value of 330 kDa at 192 h. On the contrary, the γ-PGA concentration further increased to a maximum of 30.7 g L −1 at 100 h, which is about 5 g L −1 or 20% more than in the standard approach. A positive influence of higher citric acid concentrations in medium E was also recently demonstrated by Mitsunaga et al [39]. In that work, also a higher γ-PGA concentration was reported for an increase of the citric acid concentration by the factor 1.5.…”

Section: Resultssupporting

confidence: 80%

“…3d). Acetate showed the highest concentration with 6.5 g L −1 at 192 h. The highest concentrations of acetoin, 5.2 g L −1 , and 2,3-butanediol, 1.7 g L −1 , were obtained at 120 h. Acetate, acetoin, and 2,3-butanediol are typical overflow metabolites of Bacillus species [37, 38] and have been detected as typical by-products of γ-PGA production before [31, 39]. The oxygen availability during cultivation influences the ratio of the three overflow metabolites.…”

Section: Resultsmentioning

confidence: 99%

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Investigation of poly(γ-glutamic acid) production via online determination of viscosity and oxygen transfer rate in shake flasks

et al. 2017

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BackgroundPoly(γ-glutamic acid) (γ-PGA) is a biopolymer with many useful properties making it applicable for instance in food and skin care industries, in wastewater treatment, in biodegradable plastics or in the pharmaceutical industry. γ-PGA is usually produced microbially by different Bacillus spp. The produced γ-PGA increases the viscosity of the fermentation broth. In case of shake flask fermentations, this results in an increase of the volumetric power input. The power input in shake flasks can be determined by measuring the torque of an orbitally rotating lab shaker. The online measurement of the volumetric power input enables to continuously monitor the formation or degradation of viscous products like γ-PGA. Combined with the online measurement of the oxygen transfer rate (OTR), the respiration activity of the organisms can be observed at the same time.ResultsTwo different Bacillus licheniformis strains and three medium compositions were investigated using online volumetric power input and OTR measurements as well as thorough offline analysis. The online volumetric power input measurement clearly depicted changes in γ-PGA formation due to different medium compositions as well as differences in the production behavior of the two investigated strains. A higher citric acid concentration and the addition of trace elements to the standard medium showed a positive influence on γ-PGA production. The online power input signal was used to derive an online viscosity signal which was validated with offline determined viscosity values. The online measurement of the OTR proved to be a valuable tool to follow the respiration activity of the cultivated strains and to determine its reproducibility under different cultivation conditions.ConclusionsThe combination of the volumetric power input and the OTR allows for an easy and reliable investigation of new strains, cultivation conditions and medium compositions for their potential in γ-PGA production. The power input signal and the derived online viscosity directly reflect changes in γ-PGA molecular weight and concentration, respectively, due to different cultivation conditions or production strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-017-0065-4) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Investigation of poly(γ-glutamic acid) production via online determination of viscosity and oxygen transfer rate in shake flasks

et al. 2017

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“…This results in lower metabolite concentrations for TCA cycle intermediates. These results are consistent with the metabolome analysis for γ-PGA-producing B. licheniformis (Mitsunaga et al, 2016). For higher fluxes toward γ-PGA when glycerol is used as carbon source, the precursor concentrations of citrate, isocitrate, and 2-oxoglutarate are significantly lower than for glucose with a lower γ-PGA production rate.…”

Section: Discussionsupporting

confidence: 86%

Comparison of Isomerase and Weimberg Pathway for γ-PGA Production From Xylose by Engineered Bacillus subtilis

Halmschlag

Hoffmann

Hanke

et al. 2020

Front. Bioeng. Biotechnol.

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The production of poly-γ-glutamic acid (γ-PGA), a biopolymer consisting of D-and L-glutamic acid monomers, currently relies on L-glutamate, or citrate as carbon substrates. Here we aimed at using plant biomass-derived substrates such as xylose. γ-PGA producing microorganisms including Bacillus subtilis natively metabolize xylose via the isomerase pathway. The Weimberg pathway, a xylose utilization pathway first described for Caulobacter crescentus, offers a carbon-efficient alternative converting xylose to 2-oxoglutarate without carbon loss. We engineered a recombinant B. subtilis strain that was able to grow on xylose with a growth rate of 0.43 h −1 using a recombinant Weimberg pathway. Although ion-pair reversed-phase LC/MS/MS metabolome analysis revealed lower concentrations of γ-PGA precursors such as 2-oxoglutarate, the γ-PGA titer was increased 6-fold compared to the native xylose isomerase strain. Further metabolome analysis indicates a metabolic bottleneck in the phosphoenolpyruvate-pyruvate-oxaloacetate node causing bi-phasic (diauxic) growth of the recombinant Weimberg strain. Flux balance analysis (FBA) of the γ-PGA producing B. subtilis indicated that a maximal theoretical γ-PGA yield is achieved on D-xylose/ D-glucose mixtures. The results of the B. subtilis strain harboring the Weimberg pathway on such D-xylose/ D-glucose mixtures demonstrate indeed resource efficient, high yield γ-PGA production from biomass-derived substrates.

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“…Ion-pair LC/MS/MS analysis was performed using a Nexera UHPLC system (Shimadzu, Kyoto, JAPAN) coupled with LCMS 8030 Plus (Shimadzu) as previously reported (Mitsunaga et al, 2015). The column used was a CERI (Chemicals Evaluation and Research Institute, Tokyo, JAPAN) L-column 2 ODS (150 mm×2.1 mm, particle size 3 µm).…”

Section: Ion-pair Lc/ms/ms Analysismentioning

confidence: 99%

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

Ohtake

Pontrelli

Laviña

et al. 2017

Metabolic Engineering

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A B S T R A C THigh titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15 g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3 g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies.

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Metabolome analysis reveals the effect of carbon catabolite control on the poly(γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945

Cited by 36 publications

References 35 publications

Investigation of poly(γ-glutamic acid) production via online determination of viscosity and oxygen transfer rate in shake flasks

Investigation of poly(γ-glutamic acid) production via online determination of viscosity and oxygen transfer rate in shake flasks

Comparison of Isomerase and Weimberg Pathway for γ-PGA Production From Xylose by Engineered Bacillus subtilis

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

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