The reliance of glycerol utilization by Cupriavidus necator on CO2 fixation and improved glycerol catabolism

Strittmatter, Carl Simon; Eggers, Jessica; Biesgen, Vanessa; Pauels, Inga; Becker, Florian; Steinbüchel, Alexander

doi:10.1007/s00253-022-11842-0

Cited by 12 publications

(6 citation statements)

References 72 publications

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“…In another related work, E. coli W strain underwent adaptive evolution over 1300 generations [23]. Secondly, our aALE workflow does not necessitate a genetically modified strain (e.g., the use of a mutS knock-out derivative [31]) to initiate an ALE, nor does it rely on specialized genetic tools (e.g., Tn5 mutagenesis [34]). Thirdly, our method does not depend on DNA-modifying agents, which are frequently categorized as carcinogens.…”

Section: Discussionmentioning

confidence: 99%

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Fast-track adaptive laboratory evolution ofCupriavidus necatorH16 with divalent metal cations

Sitompul,

Price,

Tee

et al. 2023

Preprint

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Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. Here, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving C. necator H16 for enhanced tolerance towards elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. Our method offers several advantages over traditional ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially hazardous DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.

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Section: Discussionmentioning

confidence: 99%

“…g ., the use of a mutS knock-out derivative [31]) to initiate an ALE, nor does it rely on specialized genetic tools ( e . g ., Tn5 mutagenesis [34]). Thirdly, our method does not depend on DNA-modifying agents, which are frequently categorized as carcinogens.…”

Section: Discussionmentioning

confidence: 99%

Fast-track adaptive laboratory evolution ofCupriavidus necatorH16 with divalent metal cations

Sitompul,

Price,

Tee

et al. 2023

Preprint

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“…For this reason, heterologous expression of light-independent enzymes in glycerol metabolism from other organism may contribute to the further improvement of biomass accumulation. Relatively better-established glycerol pathways in bacteria, hosting a similar glycerol assimilation pathway as diatom, may offer more hints [ 40 , 41 ]. Heterologous expression of GK from E. coli in Ralstonia eutropha , with important biotechnology potentials, achieved a higher glycerol uptake rate, compared to inefficient uptake in wild type due to a lower kination activity of GK [ 42 ].…”

Section: Discussionmentioning

confidence: 99%

Transcriptomic and metabolomic analyses revealed regulation mechanism of mixotrophic Cylindrotheca sp. glycerol utilization and biomass promotion

Wang

Zhou

et al. 2023

Biotechnol Biofuels

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Background Diatoms have been viewed as ideal cell factories for production of some high-value bioactive metabolites, such as fucoxanthin, but their applications are restrained by limited biomass yield. Mixotrophy, by using both CO2 and organic carbon source, is believed effective to crack the bottleneck of biomass accumulation and achieve a sustainable bioproduct supply. Results Glycerol, among tested carbon sources, was proved as the sole that could significantly promote growth of Cylindrotheca sp. with illumination, a so-called growth pattern, mixotrophy. Biomass and fucoxanthin yields of Cylindrotheca sp., grown in medium with glycerol (2 g L−1), was increased by 52% and 29%, respectively, as compared to the autotrophic culture (control) without compromise in photosynthetic performance. As Cylindrotheca sp. was unable to use glycerol without light, a time-series transcriptomic analysis was carried out to elucidate the light regulation on glycerol utilization. Among the genes participating in glycerol utilization, GPDH1, TIM1 and GAPDH1, showed the highest dependence on light. Their expressions decreased dramatically when the alga was transferred from light into darkness. Despite the reduced glycerol uptake in the dark, expressions of genes associating with pyrimidine metabolism and DNA replication were upregulated when Cylindrotheca sp. was cultured mixotrophically. Comparative transcriptomic and metabolomic analyses revealed amino acids and aminoacyl-tRNA metabolisms were enhanced at different timepoints of diurnal cycles in mixotrophic Cylindrotheca sp., as compared to the control. Conclusions Conclusively, this study not only provides an alternative for large-scale cultivation of Cylindrotheca, but also pinpoints the limiting enzymes subject to further metabolic manipulation. Most importantly, the novel insights in this study should aid to understand the mechanism of biomass promotion in mixotrophic Cylindrotheca sp.

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“…Meanwhile, it has been suggested that R. eutropha can fix carbon dioxide even under heterotrophic culture conditions [43]. Furthermore, carbon dioxide fixation appears to be promoted by the presence of glycerol, a substrate for R. eutropha that is hardly assimilated under heterotrophic culture conditions [44][45][46][47]. In this study, a high P(3HB) yield of 0.28 g-polymer/g-glucose was observed when R. eutropha NCIMB 11599 was cultured in MS medium supplemented with 20 g/L 13 C-glucose.…”

Section: Discussionmentioning

confidence: 99%

Poly(3-Hydroxybutyrate) Biosynthesis from [U-13C6]D-Glucose by Ralstonia eutropha NCIMB 11599 and Recombinant Escherichia coli

Sivashankari,

Miyahara,

Tsuge

2023

Microbiology Research

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The use of stable isotope-labeled polymers in in situ biodegradation tests provides detailed information on the degradation process. As isotope-labeled raw chemicals are generally expensive, it is desirable to prepare polymer samples with high production yields and high isotope-labeling ratios. The biodegradable plastic poly[(R)-3-hydroxybutyrate)] (P(3HB)) is produced by microorganisms. In this study, to produce carbon 13 (13C)-labeled P(3HB) from [U-13C6]D-glucose (13C-glucose), the culture conditions needed for high production yields and high 13C-labeling ratios were investigated using Ralstonia eutropha NCIMB 11599 and recombinant Escherichia coli JM109. We found that over 10 g/L of P(3HB) could be obtained when these microorganisms were cultured in Luria-Bertani (LB3) medium containing 3 g/L NaCl and 40 g/L 13C-glucose, while 1.4–4.7 g/L of P(3HB) was obtained when a mineral salt (MS) medium containing 20 g/L 13C-glucose was used. The 13C-labeling ratio of P(3HB) was determined by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry (GC-MS), and both analytical methods yielded nearly identical results. High 13C-labeling ratios (97.6 atom% by GC-MS) were observed in the MS medium, whereas low 13C-labeling ratios (88.8–94.4 atom% by GC-MS) were observed in the LB3 medium. Isotope effects were observed for the P(3HB) content in cells cultured in the LB3 medium and the polydispersity of P(3HB).

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The reliance of glycerol utilization by Cupriavidus necator on CO2 fixation and improved glycerol catabolism

Cited by 12 publications

References 72 publications

Fast-track adaptive laboratory evolution ofCupriavidus necatorH16 with divalent metal cations

Fast-track adaptive laboratory evolution ofCupriavidus necatorH16 with divalent metal cations

Transcriptomic and metabolomic analyses revealed regulation mechanism of mixotrophic Cylindrotheca sp. glycerol utilization and biomass promotion

Poly(3-Hydroxybutyrate) Biosynthesis from [U-13C6]D-Glucose by Ralstonia eutropha NCIMB 11599 and Recombinant Escherichia coli

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