scholarly journals Tung Oil-Based Production of High 3-Hydroxyhexanoate-Containing Terpolymer Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate-co-3-Hydroxyhexanoate) Using Engineered Ralstonia eutropha

Polymers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1084
Author(s):  
Hye Soo Lee ◽  
Sun Mi Lee ◽  
Sol Lee Park ◽  
Tae-Rim Choi ◽  
Hun-Suk Song ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Eleostearic Acid ◽  
Fossil Fuels ◽  
Ralstonia Eutropha ◽  
Wild Type ◽  
Medical Field ◽  
Tung Oil ◽  
Medium Chain Length ◽  
Partial Harvesting ◽  
Biodegradable Properties

Polyhydroxyalkanoates (PHAs) are attractive new bioplastics for the replacement of plastics derived from fossil fuels. With their biodegradable properties, they have also recently been applied to the medical field. As poly(3-hydroxybutyrate) produced by wild-type Ralstonia eutropha has limitations with regard to its physical properties, it is advantageous to synthesize co- or terpolymers with medium-chain-length monomers. In this study, tung oil, which has antioxidant activity due to its 80% α-eleostearic acid content, was used as a carbon source and terpolymer P(53 mol% 3-hydroxybytyrate-co-2 mol% 3-hydroxyvalerate-co-45 mol% 3-hydroxyhexanoate) with a high proportion of 3-hydroxyhexanoate was produced in R. eutropha Re2133/pCB81. To avail the benefits of α-eleostearic acid in the tung oil-based medium, we performed partial harvesting of PHA by using a mild water wash to recover PHA and residual tung oil on the PHA film. This resulted in a film coated with residual tung oil, showing antioxidant activity. Here, we report the first application of tung oil as a substrate for PHA production, introducing a high proportion of hydroxyhexanoate monomer into the terpolymer. Additionally, the residual tung oil was used as an antioxidant coating, resulting in the production of bioactive PHA, expanding the applicability to the medical field.

scholarly journals Untargeted metabolomics analysis of Ralstonia eutropha during plant oil cultivations reveals the presence of a fucose salvage pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Björn Gutschmann ◽  
Martina C. E. Bock ◽  
Stefan Jahns ◽  
Peter Neubauer ◽  
Christopher J. Brigham ◽  
...  
Keyword(s):  
De Novo ◽  
Ralstonia Eutropha ◽  
Wild Type ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Plant Oil ◽  
Microbial Physiology ◽  
Medium Chain Length ◽  
Metabolic States ◽  
Metabolomic Study

AbstractProcess engineering of biotechnological productions can benefit greatly from comprehensive analysis of microbial physiology and metabolism. Ralstonia eutropha (syn. Cupriavidus necator) is one of the best studied organisms for the synthesis of biodegradable polyhydroxyalkanoate (PHA). A comprehensive metabolomic study during bioreactor cultivations with the wild-type (H16) and an engineered (Re2058/pCB113) R. eutropha strain for short- and or medium-chain-length PHA synthesis has been carried out. PHA production from plant oil was triggered through nitrogen limitation. Sample quenching allowed to conserve the metabolic states of the cells for subsequent untargeted metabolomic analysis, which consisted of GC–MS and LC–MS analysis. Multivariate data analysis resulted in identification of significant changes in concentrations of oxidative stress-related metabolites and a subsequent accumulation of antioxidative compounds. Moreover, metabolites involved in the de novo synthesis of GDP-l-fucose as well as the fucose salvage pathway were identified. The related formation of fucose-containing exopolysaccharides potentially supports the emulsion-based growth of R. eutropha on plant oils.

scholarly journals Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation

2020 ◽  
Vol 22 (1) ◽  
pp. 152
Author(s):  
Dorota Dabrowska ◽  
Justyna Mozejko-Ciesielska ◽  
Tomasz Pokój ◽  
Slawomir Ciesielski
Keyword(s):  
Chain Length ◽  
Nitrogen Limitation ◽  
Stringent Response ◽  
Wild Type ◽  
Pseudomonas Putida Kt2440 ◽  
Medium Chain ◽  
Environmental Stimuli ◽  
Medium Chain Length ◽  
In The Wild

Pseudomonas putida’s versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs’ metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 825-833 ◽  
Author(s):  
Markus Pötter ◽  
Helena Müller ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Current Model ◽  
Transcriptional Repressor ◽  
Start Codon ◽  
Wild Type ◽  
Mobility Shift ◽  
Intergenic Regions ◽  
Gel Mobility Shift ◽  
Dnasei Footprinting ◽  
Similar Growth

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123 and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3 and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1 mutant and the ΔphaP12, ΔphaP123 and ΔphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3 or ΔphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

scholarly journals Versatile Metabolic Adaptations ofRalstonia eutrophaH16 to a Loss of PdhL, the E3 Component of the Pyruvate Dehydrogenase Complex

2011 ◽  
Vol 77 (7) ◽  
pp. 2254-2263 ◽  
Author(s):  
Matthias Raberg ◽  
Jan Bechmann ◽  
Ulrike Brandt ◽  
Jonas Schlüter ◽  
Bianca Uischner ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Burkholderia Cepacia ◽  
Ralstonia Eutropha ◽  
Pyruvate Dehydrogenase Complex ◽  
Synthesis Rate ◽  
Wild Type ◽  
Dimerization Domain ◽  
Amino Terminal ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

ABSTRACTA previous study reported that the Tn5-induced poly(3-hydroxybutyric acid) (PHB)-leaky mutantRalstonia eutrophaH1482 showed a reduced PHB synthesis rate and significantly lower dihydrolipoamide dehydrogenase (DHLDH) activity than the wild-typeR. eutrophaH16 but similar growth behavior. Insertion of Tn5was localized in thepdhLgene encoding the DHLDH (E3 component) of the pyruvate dehydrogenase complex (PDHC). Taking advantage of the available genome sequence ofR. eutrophaH16, observations were verified and further detailed analyses and experiments were done.In silicogenome analysis revealed thatR. eutrophapossesses all five known types of 2-oxoacid multienzyme complexes and five DHLDH-coding genes. Of these DHLDHs, only PdhL harbors an amino-terminal lipoyl domain. Furthermore, insertion of Tn5inpdhLof mutant H1482 disrupted the carboxy-terminal dimerization domain, thereby causing synthesis of a truncated PdhL lacking this essential region, obviously leading to an inactive enzyme. The defined ΔpdhLdeletion mutant ofR. eutrophaexhibited the same phenotype as the Tn5mutant H1482; this excludes polar effects as the cause of the phenotype of the Tn5mutant H1482. However, insertion of Tn5or deletion ofpdhLdecreases DHLDH activity, probably negatively affecting PDHC activity, causing the mutant phenotype. Moreover, complementation experiments showed that different plasmid-encoded E3 components ofR. eutrophaH16 or of other bacteria, likeBurkholderia cepacia, were able to restore the wild-type phenotype at least partially. Interestingly, the E3 component ofB. cepaciapossesses an amino-terminal lipoyl domain, like the wild-type H16. A comparison of the proteomes of the wild-type H16 and of the mutant H1482 revealed striking differences and allowed us to reconstruct at least partially the impressive adaptations ofR. eutrophaH1482 to the loss of PdhL on the cellular level.

scholarly journals PO-191 The effect of hypoxic training at different simulated altitude on the antioxidant activity mediated by Nrf2 in mice skeletal muscle

2018 ◽  
Vol 1 (4) ◽  
Author(s):  
Weixiu Ji ◽  
Ying Zhang
Keyword(s):  
Skeletal Muscle ◽  
Antioxidant Activity ◽  
Oxygen Concentration ◽  
Training Group ◽  
Control Group ◽  
Simulated Altitude ◽  
Wild Type ◽  
High Quality ◽  
Hypoxic Training ◽  
Nrf2 Protein

Objective It is generally believed that the long-term hypoxic training could impact oxidation resistance. Nrf2-Keapl signaling pathway is a key pathway of cell oxidative stress reaction. This research attempts to investigate the role and mechanism of Nrf2 in oxidation resistance to hypoxic training of different oxygen concentration. Methods Part one, 8-week-old Nrf2 knockout mice and wild type mice were divided into normoxic control group (NC), simulated altitude of 3500m hypoxic training group (3500HT) and simulated altitude of 5000m hypoxic training group (5000HT) randomly and respectively. The mice run on treadmill in speed of 12 m/min, 1h/day, 6day/week, for 4 weeks. Oxygen concentration in hypoxia was 13.3% and 10%. Mice were treated for 4 weeks, 8h/day. 48 h after the last training, the mice were sacrificed and skeletal muscles of legs were collected. Western Blot tested Nrf2 and antioxidant enzyme protein. Antioxidant enzymes mRNA were tested by RT-PCR. High quality fluorescence measurement was used to test ROS levels in skeletal muscle. Part two, The 30 C57BL/6J mice were divided into three groups: control group (WC), hypoxia group (WH), hypoxic training group (WHT). The hypoxic training arrangement was same as before. After both the interventions, the mice were sacrificed and collected skeletal muscle of legs. The expression of Nrf2, Keap1 and p-Nrf2 were analyzed by western blot. High quality fluorescence assay was done to detect ROS level in skeletal muscle of mice. Results (1) Compared with the same type mice NC group, Nrf2 protein, the mRNA and protein of CAT, GPX-1, GCLm, the mRNA of SOD1, SOD2, HO-1 were increased in wild type mice 3500HT group. And the Nrf2 protein, the mRNA and protein of SOD1, SOD2, the mRNA of CAT, NQO-1, GCLc, GCLm mRNA, the protein of HO-1 were decreased, and the ROS levels was higher in wile type mice 5000HT group. The mRNA of CAT, HO-1 in Nrf2-KO mice 3500HT group were increased, the mRNA and protein of SOD1, the mRNA of SOD2, the protein of GCLc were decreased, but the GCLc mRNA was increased in Nrf2-KO mice 5000HT group. When compared with the same intervention wild type mice, the mRNA and protein of SOD1, GPX-1, SOD2, HO-1, the mRNA of CAT, NQO-1, GCLc, GCLm were decreased in Nrf2-KO mice 3500HT group. The mRNA of GCLm, NQO-1, the protein of GCLc, HO-1 were decreased, but the GCLc mRNA was increased. (2) Nrf2/Keap1 complex contents in mice skeletal muscle of WH and WHT groups were significantly increased compared with WC group respectively. The free Nrf2 in mice skeletal muscle of WH、WHT groups were significantly reduced compared with WC group respectively. After both types of intervention, free Keap1 had no change nearly in skeletal muscle of mice. Compared with WC group, p-Nrf2 in mice skeletal muscle of WH and WHT groups were significantly reduced. The ROS level in mice skeletal muscle of WHT group significantly increased compared with WC group mice. Conclusions: Hypoxia and hypoxia training three interventions could increase Nrf2/Keap1 combination in skeletal muscle of mice, reduce the volume of free Nrf2; Phosphorylation of Nrf2 in skeletal muscle of mice in hypoxia training group was significantly lower, which may be result in marked increase in ROS level. Conclusions (1) Hypoxic training could affect antioxidant activity via Nrf2 in mice skeletal muscle, which is connected with the oxygen concentration. (2) Moderate hypoxia training (at the altitude of 3500m) can promote the antioxidant activity via Nrf2. However, extremely hypoxic training (at the altitude of 5000m) can restrain the antioxidant activity via Nrf2 through the inhibition of Nrf2/Keap1 dissociation.  

scholarly journals A DNA Region Recognized by the Nitric Oxide-Responsive Transcriptional Activator NorR Is Conserved in β- and γ-Proteobacteria

2004 ◽  
Vol 186 (23) ◽  
pp. 7980-7987 ◽  
Author(s):  
Andrea Büsch ◽  
Anne Pohlmann ◽  
Bärbel Friedrich ◽  
Rainer Cramm
Keyword(s):  
Nitric Oxide ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Ralstonia Eutropha ◽  
Upstream Region ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Genome Database ◽  
Nitric Oxide Metabolism ◽  
Signaling Domain

ABSTRACT The σ54-dependent regulator NorR activates transcription of target genes in response to nitric oxide (NO) or NO-generating agents. In Ralstonia eutropha H16, NorR activates transcription of the dicistronic norAB operon that encodes NorA, a protein of unknown function, and NorB, a nitric oxide reductase. A constitutively activating NorR derivative (NorR′), in which the N-terminal signaling domain was replaced by MalE, specifically bound to the norAB upstream region as revealed by gel retardation analysis. Within a 73-bp DNA segment protected by MalE-NorR′ in a DNase I footprint assay, three conserved inverted repeats, GGT-(N7)-ACC (where N is any base), that we consider to be NorR-binding boxes were identified. Mutations altering the spacing or the base sequence of these repeats resulted in an 80 to 90% decrease of transcriptional activation by wild-type NorR. Genome database analyses demonstrate that the GT-(N7)-AC core of the inverted repeat is found in several proteobacteria upstream of gene loci encoding proteins of nitric oxide metabolism, including nitric oxide reductase (NorB), flavorubredoxin (NorV), NO dioxygenase (Hmp), and hybrid cluster protein (Hcp).

Analysis of the Seed Lipids of Aleurites montana

1998 ◽  
Vol 53 (5-6) ◽  
pp. 305-310 ◽  
Author(s):  
A. Radunz ◽  
P. Heb ◽  
G. H. Schmid
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Silica Gel ◽  
Eleostearic Acid ◽  
Saturated Fatty Acids ◽  
Tung Oil ◽  
Seed Lipids

Abstract The seed lipids of the Chinese tung-oil tree Aleurites montana are composed of 97.2% triglycerides, 2% phospholipids and of 0.8% glycolipids. In the triglycerides 67% of all fatty acids are α-eleostearic acid (configuration C18:3, ∆9 cis, ∆11 trans, ∆13 trans). This acid is bound in the triglycerides in the 1,3 position. Via mercury Il-acetate adducts and subsequent chromatography on silica gel a separation of this fatty acid from the seed fatty acids is possible. By this procedure an a-eleostearic acid-free tung-oil mixture is obtained, which consists by one half of C18:2, 25% of C18:1 and by 25% of the saturated fatty acids C16:0, C18:0 and C14:0.

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures

2018 ◽  
Vol 28 (5) ◽  
pp. 225-235
Author(s):  
Edmar Ramos Oliveira-Filho ◽  
Linda P. Guamán ◽  
Thatiane Teixeira Mendonça ◽  
Paul F. Long ◽  
Marilda Keico Taciro ◽  
...  
Keyword(s):  
Carbon Source ◽  
Aeromonas Hydrophila ◽  
Sole Carbon Source ◽  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Medium Chain Length ◽  
Marker Free ◽  
Pha Copolymers ◽  
First Time ◽  
Induced Mutant

Three different polyhydroxyalkanoate (PHA) synthase genes (<i>Ralstonia eutropha</i> H16, <i>Aeromonas</i> sp. TSM81 or <i>Aeromonas hydrophila</i> ATCC7966 <i>phaC</i>) were introduced into the chromosome of two <i>Pseudomonas</i> strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHA<sub>MCL</sub>) producer (<i>Pseudomonas</i> sp. LFM046) and a UV-induced mutant strain unable to produce PHA (<i>Pseudomonas</i> sp. LFM461). We reported for the first time the insertion of a chromosomal copy of <i>phaC</i> using the transposon system mini-Tn<i>7</i>. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-<i>co</i>-3HA<sub>MCL</sub>) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring <i>A. hydrophila phaC</i> produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of <i>Pseudomonas</i> sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HA<sub>MCL</sub> using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

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