scholarly journals Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

2011 ◽  
Vol 77 (9) ◽  
pp. 2847-2854 ◽  
Author(s):  
Charles F. Budde ◽  
Sebastian L. Riedel ◽  
Laura B. Willis ◽  
ChoKyun Rha ◽  
Anthony J. Sinskey
Keyword(s):  
Palm Oil ◽  
Ralstonia Eutropha ◽  
Plasmid Stability ◽  
Dry Weight ◽  
Pha Synthase ◽  
Plant Oil ◽  
Short Chain Length ◽  
Recombinant Gene Expression ◽  
Content Type ◽  
Coa Reductase

ABSTRACTThe polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinantRalstonia eutrophastrains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered twoR. eutrophastrains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacteriumRhodococcus aetherivoransI24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) fromPseudomonas aeruginosawas shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded byphaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so anR. eutrophaplasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase geneproCwas deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.

scholarly journals From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

2013 ◽  
Vol 79 (12) ◽  
pp. 3582-3589 ◽  
Author(s):  
Daniel Heinrich ◽  
Björn Andreessen ◽  
Mohamed H. Madkour ◽  
Mansour A. Al-Ghamdi ◽  
Ibrahim I. Shabbaj ◽  
...  
Keyword(s):  
Carbon Source ◽  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Biodiesel Production ◽  
Pha Synthase ◽  
Content Type ◽  
Coa Transferase ◽  
Fed Batch Fermentation ◽  
A Cell ◽  
Cultivation Process

ABSTRACTIn recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) ofPseudomonas putidaKT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) ofClostridium propionicumX2, and polyhydroxyalkanoate (PHA) synthase (phaC1) ofRalstonia eutrophaH16 were cloned and expressed in the 1,3-propanediol producerShimwellia blattae. In a two-step cultivation process, recombinantS. blattaecells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.

scholarly journals Identification and Analysis of the Polyhydroxyalkanoate-Specific β-Ketothiolase and Acetoacetyl Coenzyme A Reductase Genes in the Cyanobacterium Synechocystis sp. Strain PCC6803

2000 ◽  
Vol 66 (10) ◽  
pp. 4440-4448 ◽  
Author(s):  
Gaspar Taroncher-Oldenburg ◽  
Koren Nishina ◽  
Gregory Stephanopoulos
Keyword(s):  
Sequence Analysis ◽  
Coenzyme A ◽  
Dry Weight ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Pha Synthase ◽  
Promoter Regions ◽  
Short Chain Length ◽  
Heterologous Transformation ◽  
Coa Reductase

ABSTRACT Synechocystis sp. strain PCC6803 possesses a polyhydroxyalkanoate (PHA)-specific β-ketothiolase encoded byphaASyn and an acetoacetyl-coenzyme A (CoA) reductase encoded by phaBSyn . A similarity search of the entire Synechocystis genome sequence identified a cluster of two putative open reading frames (ORFs) for these genes, slr1993 and slr1994. Sequence analysis showed that the ORFs encode proteins having 409 and 240 amino acids, respectively. The two ORFs are colinear and most probably coexpressed, as revealed by sequence analysis of the promoter regions. Heterologous transformation of Escherichia coli with the two genes and the PHA synthase of Synechocystis resulted in accumulation of PHAs that accounted for up to 12.3% of the cell dry weight under high-glucose growth conditions. Targeted disruption of the above gene cluster inSynechocystis eliminated the accumulation of PHAs. ORFs slr1993 and slr1994 thus encode the PHA-specific β-ketothiolase and acetoacetyl-CoA reductase of Synechocystis and, together with the recently characterized PHA synthase genes in this organism (S. Hein, H. Tran, and A. Steinbüchel, Arch. Microbiol. 170:162–170, 1998), form the first complete PHA biosynthesis pathway known in cyanobacteria. Sequence alignment of all known short-chain-length PHA-specific acetoacetyl-CoA reductases also suggests an extended signature sequence, VTGXXXGIG, for this group of proteins. Phylogenetic analysis further places the origin ofphaASyn and phaBSyn in the γ subdivision of the division Proteobacteria.

scholarly journals Rearrangement of Gene Order in thephaCABOperon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[(R)-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli

2012 ◽  
Vol 78 (9) ◽  
pp. 3177-3184 ◽  
Author(s):  
Ayaka Hiroe ◽  
Kenji Tsuge ◽  
Christopher T. Nomura ◽  
Mitsuhiro Itaya ◽  
Takeharu Tsuge
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gene Order ◽  
Ralstonia Eutropha ◽  
Genetically Engineered ◽  
Pha Synthase ◽  
Limiting Factor ◽  
Expression Plasmid ◽  
Content Type ◽  
Ultrahigh Molecular Weight

ABSTRACTUltrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineeredEscherichia coliis an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized asphaCAB) encodes the essential proteins for the production of P(3HB) in the native producer,Ralstonia eutropha. The three genes of thephaCABoperon arephaC, which encodes the polyhydroxyalkanoate (PHA) synthase,phaA, which encodes a 3-ketothiolase, andphaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of thephaCABoperon (phaABC,phaACB,phaBAC,phaBCA,phaCAB, andphaCBA) on an expression plasmid in genetically engineeredE. coliwas examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of thephagenes relative to the promoter. The most balanced production result was achieved in the strain harboring thephaBCAexpression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.

scholarly journals Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

2014 ◽  
Vol 80 (24) ◽  
pp. 7702-7709 ◽  
Author(s):  
Jessica Eggers ◽  
Alexander Steinbüchel
Keyword(s):  
Escherichia Coli ◽  
Ralstonia Eutropha ◽  
Model Organism ◽  
Dry Weight ◽  
Growth Behavior ◽  
Carbon Limitation ◽  
Content Type ◽  
Phb Depolymerase ◽  
E Coli ◽  
The Impact

ABSTRACTThe model organism for polyhydroxybutyrate (PHB) biosynthesis,Ralstonia eutrophaH16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinantEscherichia coliBL21(DE3) strains were used to study the impact of selected PHB depolymerases ofR. eutrophaH16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinantE. coliBL21(DE3) strains were constructed, which harbored a plasmid carrying thephaCABoperon fromR. eutrophaH16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase fromR. eutrophaH16. It is shown in this study that the growth behavior of the respective recombinantE. colistrains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboringphaZ7reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed ifphaZ1was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.

scholarly journals Altering the Substrate Specificity of Polyhydroxyalkanoate Synthase 1 Derived from Pseudomonas putida GPo1 by Localized Semirandom Mutagenesis

2004 ◽  
Vol 186 (13) ◽  
pp. 4177-4184 ◽  
Author(s):  
Der-Shyan Sheu ◽  
Chia-Yin Lee
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Chain Length ◽  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Multiple Point ◽  
Evolution System ◽  
Degenerate Primers ◽  
Short Chain Length ◽  
Single Polymer Chain

ABSTRACT The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 Pp , class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA− as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB−4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB−4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB−4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.

scholarly journals Employing a Recombinant Strain of Advenella mimigardefordensis for Biotechnical Production of Homopolythioesters from 3,3′-Dithiodipropionic Acid

2012 ◽  
Vol 78 (9) ◽  
pp. 3286-3297 ◽  
Author(s):  
Yongzhen Xia ◽  
Jan Hendrik Wübbeler ◽  
Qingsheng Qi ◽  
Alexander Steinbüchel
Keyword(s):  
Carbon Source ◽  
Dry Weight ◽  
Pha Synthase ◽  
Mineral Salts ◽  
Content Type ◽  
Genes Encoding ◽  
Polyhydroxyalkanoate Synthase ◽  
Encoding Gene ◽  
Different Origins ◽  
Severe Growth

ABSTRACTAdvenella mimigardefordensisstrain DPN7Twas genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3′-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of thebuk-ptboperon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, fromClostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaCAm). These measures yielded the potent PMP production strainA. mimigardefordensisstrain SHX22. The deletion ofmdowas required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-bornebuk-ptboperon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase ofA. mimigardefordensis(phaCAm) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate.

scholarly journals Effects of Homologous Phosphoenolpyruvate-Carbohydrate Phosphotransferase System Proteins on Carbohydrate Uptake and Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha H16

2011 ◽  
Vol 77 (11) ◽  
pp. 3582-3590 ◽  
Author(s):  
Chlud Kaddor ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Insertion Site ◽  
Carbon Sources ◽  
Sugar Transport ◽  
Dry Weight ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Homologous Proteins ◽  
Content Type ◽  
Negative Effect

ABSTRACTSeven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome ofRalstonia eutrophaH16 byin silicoanalysis. Except theN-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ΔfrcACBand H16 ΔnagFECmutants exhibited no growth when cultivated on fructose andN-acetylglucosamine, respectively. Furthermore, a transposon mutant with aptsM-ptsHinsertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mobinsertion.ptsM,ptsH, andptsIsingle, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384,frcACB, ornagFECgenes. In contrast, mutant H16 ΔH16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of theptsMHIdeletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins inR. eutrophaH16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism.

scholarly journals In-Line Monitoring of Polyhydroxyalkanoate (PHA) Production during High-Cell-Density Plant Oil Cultivations Using Photon Density Wave Spectroscopy

2019 ◽  
Vol 6 (3) ◽  
pp. 85 ◽  
Author(s):  
Björn Gutschmann ◽  
Thomas Schiewe ◽  
Manon T.H. Weiske ◽  
Peter Neubauer ◽  
Roland Hass ◽  
...  
Keyword(s):  
Cell Density ◽  
Ralstonia Eutropha ◽  
High Cell Density ◽  
Process Analytical Technology ◽  
Density Wave ◽  
Dry Weight ◽  
Photon Density ◽  
High Cell ◽  
Plant Oil ◽  
Pha Production

Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient µs’ and the absorption coefficient µa at 638 nm. A correlation of µs’ with the cell dry weight (CDW) and µa with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse feed cultivations. The correlation was used to predict CDW, RCDW, and PHB formation in a high-cell-density fed-batch cultivation with a productivity of 1.65 gPHB·L−1·h−1 and a final biomass of 106 g·L−1 containing 73 wt% PHB. The new method applied in this study allows in-line monitoring of CDW, RCDW, and PHA formation.

scholarly journals Autotrophic Production of Stable-Isotope-Labeled Arginine in Ralstonia eutropha Strain H16

2012 ◽  
Vol 78 (22) ◽  
pp. 7884-7890 ◽  
Author(s):  
Steffen Lütte ◽  
Anne Pohlmann ◽  
Evgeny Zaychikov ◽  
Edward Schwartz ◽  
Johannes R. Becher ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Ralstonia Eutropha ◽  
Expression System ◽  
Maximum Yield ◽  
Substantial Reduction ◽  
Dry Weight ◽  
Growth Conditions ◽  
Scale Production ◽  
Content Type ◽  
Fed Batch Fermentation

ABSTRACTWith the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains ofRalstonia eutrophaunder lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) ofSynechocystissp. strain PCC6308 under the control of the strong PcbbLpromoter of theR. eutrophaH16cbbcoperon (coding for autotrophic CO2fixation). In batch cultures growing on H2and CO2as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression ofcphAduring lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with13CO2and15NH4Cl, and the13C/15N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [13C/15N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%.

scholarly journals Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics

2013 ◽  
Vol 79 (19) ◽  
pp. 6134-6139 ◽  
Author(s):  
Ken'ichiro Matsumoto ◽  
Yoshikazu Tanaka ◽  
Tsuyoshi Watanabe ◽  
Ren Motohashi ◽  
Koji Ikeda ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Directed Evolution ◽  
Coenzyme A ◽  
Ralstonia Eutropha ◽  
Wild Type ◽  
Content Type ◽  
Enhanced Activity ◽  
Coa Reductase ◽  
Acetoacetyl Coa Reductase

ABSTRACTNADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB fromRalstonia eutrophawas engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-basedin vivoscreening system usingEscherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibitedkcatvalues that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinantCorynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

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