scholarly journals Accumulation of the PhaP Phasin of Ralstonia eutropha Is Dependent on Production of Polyhydroxybutyrate in Cells

2001 ◽  
Vol 183 (14) ◽  
pp. 4217-4226 ◽  
Author(s):  
Gregory M. York ◽  
Björn H. Junker ◽  
JoAnne Stubbe ◽  
Anthony J. Sinskey
Keyword(s):  
Regulatory Mechanism ◽  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Gene Replacement ◽  
Pha Synthase ◽  
Wild Type ◽  
Phb Production ◽  
Associated Proteins ◽  
Mixed Populations ◽  
In Cells

ABSTRACT Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and aphaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of thephaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.

scholarly journals The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

2002 ◽  
Vol 184 (1) ◽  
pp. 59-66 ◽  
Author(s):  
Gregory M. York ◽  
JoAnne Stubbe ◽  
Anthony J. Sinskey
Keyword(s):  
Ralstonia Eutropha ◽  
Regulatory System ◽  
Negative Regulator ◽  
Pha Synthase ◽  
Deletion Strain ◽  
Wild Type ◽  
Heterologous System ◽  
Pathway Models ◽  
Pha Production ◽  
In Cells

ABSTRACT Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.

scholarly journals Impact of Multiple β-Ketothiolase Deletion Mutations in Ralstonia eutropha H16 on the Composition of 3-Mercaptopropionic Acid-Containing Copolymers

2010 ◽  
Vol 76 (16) ◽  
pp. 5373-5382 ◽  
Author(s):  
Nicole Lindenkamp ◽  
Katja Peplinski ◽  
Elena Volodina ◽  
Armin Ehrenreich ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Deletion Mutants ◽  
Transcriptome Data ◽  
Wild Type ◽  
Acetyl Coenzyme A ◽  
Deletion Mutations ◽  
Knockout Mutants ◽  
In The Wild ◽  
Percentage Ratio

ABSTRACT β-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] synthesis in bacteria by condensing two molecules of acetyl coenzyme A (acetyl-CoA) to acetoacetyl-CoA. Analyses of the genome sequence of Ralstonia eutropha H16 revealed 15 isoenzymes of PhaA in this bacterium. In this study, we generated knockout mutants of various phaA homologues to investigate their role in and contributions to poly(3HB) metabolism and to suppress biosynthesis of 3HB-CoA for obtaining enhanced molar 3-mercaptopriopionate (3MP) contents in poly(3HB-co-3MP) copolymers when cells were grown on gluconate plus 3-mercaptopropionate or 3,3′-dithiodipropionate. In silico sequence analysis of PhaA homologues, transcriptome data, and other aspects recommended the homologues phaA, bktB, H16_A1713/H16_B1771, H16_A1528, H16_B1369, H16_B0381, and H16_A0170 for further analysis. Single- and multiple-deletion mutants were generated to investigate the influence of these β-ketothiolases on growth and polymer accumulation. The deletion of single genes resulted in no significant differences from the wild type regarding growth and polymer accumulation during cultivation on gluconate or gluconate plus 3MP. Deletion of phaA plus bktB (H16Δ2 mutant) resulted in approximately 30% less polymer accumulation than in the wild type. Deletion of H16_A1713/H16_B1771, H16_A1528, H16_B0381, and H16_B1369 in addition to phaA and bktB gave no differences in comparison to the H16Δ2 mutant. In contrast, deletion of H16_A0170 additionally to phaA and bktB yielded a mutant which accumulated about 30% poly(3HB) (wt/wt of the cell dry weight [CDW]). Although we were not able to suppress poly(3HB) biosynthesis completely, the copolymer compositions could be altered significantly with a lowered percentage ratio of 3HB constituents (from 85 to 52 mol%) and an increased percentage ratio of 3MP constituents (from 15 to 48 mol%), respectively. In this study, we demonstrated that PhaA, BktB, and H16_A0170 are majorly involved in poly(3HB) synthesis in R. eutropha H16. A fourth β-ketothiolase or a combination of several of the other β-ketothiolases contributed to a maximum of only 30% (wt/wt of CDW) of the remaining (co)polymer.

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 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 Ralstonia eutropha H16 Encodes Two and Possibly Three Intracellular Poly[d-(−)-3-Hydroxybutyrate] Depolymerase Genes

2003 ◽  
Vol 185 (13) ◽  
pp. 3788-3794 ◽  
Author(s):  
Gregory M. York ◽  
Joachim Lupberger ◽  
Jiamin Tian ◽  
Adam G. Lawrence ◽  
JoAnne Stubbe ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Complete Sequence ◽  
Inverse Pcr ◽  
Degenerate Primers ◽  
Phb Depolymerase ◽  
Phb Production ◽  
Ralstonia Metallidurans

ABSTRACT Intracellular poly[d-(−)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share ∼30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (ΔphaZ1, ΔphaZ2, ΔphaZ3, ΔphaZ1ΔphaZ2, ΔphaZ1ΔphaZ3, ΔphaZ2ΔphaZ3, and ΔphaZ1ΔphaZ2ΔphaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated ∼80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.

scholarly journals Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4897-4903 ◽  
Author(s):  
Jong-il Choi ◽  
Sang Yup Lee ◽  
Kyuboem Han
Keyword(s):  
Escherichia Coli ◽  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Wild Type ◽  
High Productivity ◽  
E Coli ◽  
Alcaligenes Latus ◽  
Enhanced Production ◽  
Wide Range ◽  
High Level

ABSTRACT Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. RecombinantEscherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinantE. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. RecombinantE. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinantE. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.

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 Conversion of Glycerol to Poly(3-Hydroxypropionate) in Recombinant Escherichia coli

2009 ◽  
Vol 76 (2) ◽  
pp. 622-626 ◽  
Author(s):  
Björn Andreeßen ◽  
Alvin Brian Lange ◽  
Horst Robenek ◽  
Alexander Steinbüchel
Keyword(s):  
Escherichia Coli ◽  
Batch Fermentation ◽  
Ralstonia Eutropha ◽  
Dry Weight ◽  
Recombinant Escherichia Coli ◽  
Pha Synthase ◽  
Fed Batch ◽  
Interesting Application ◽  
Serovar Typhimurium ◽  
Fed Batch Fermentation

ABSTRACT We have developed the conversion of glycerol into thermoplastic poly(3-hydroxypropionate) [poly(3HP)]. For this, the genes for glycerol dehydratase (dhaB1) of Clostridium butyricum, propionaldehyde dehydrogenase (pduP) of Salmonella enterica serovar Typhimurium LT2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha were expressed in recombinant Escherichia coli. Poly(3HP) was accumulated up to 11.98% (wt/wt [cell dry weight]) in a two-step, fed-batch fermentation. The present study shows an interesting application to engineer a poly(3HP) synthesis pathway in bacteria.

scholarly journals Technical-Economical Approach for PHB Production by Ralstonia Eutropha Strain Using Concentrated Vinasse as Carbon Source and Other Biotechnological Applications

2021 ◽  
Author(s):  
Manuella Silverio ◽  
Rosane Piccoli ◽  
João Reis ◽  
José Gregorio Gomez ◽  
Antonio Baptista
Keyword(s):  
Carbon Source ◽  
Ralstonia Eutropha ◽  
Culture Media ◽  
Dry Weight ◽  
Global Market ◽  
Cellular Growth ◽  
Production Costs ◽  
E Coli ◽  
Phb Production ◽  
Threonine Production

Abstract The Brazilian ethanol industry is one of the most important in the global market, however these important industrial activities have been generating significant amounts of vinasse and its management has become costly for distilleries. In this study, the aim was to evaluate concentrated and in natura vinasse as basal culture media for biotechnological processes. Different bacteria and processes were assessed: L-threonine production by E. coli THR14, with glucose as carbon source; PHB production by halophilic strain Halomonas sp. HG03, with sucrose as carbon source; and PHB biosynthesis by R. eutropha L359PCJ, which used glycerol from vinasse as carbon source. Strains were evaluated firstly in shake flasks cultivations using vinasse-based media. E. coli THR14 had no statistical difference for biomass and L-threonine concentrations among control and vinasse-based treatments (up to 50% v v-1 of in natura vinasse). Halomonas sp. HG03 and R. eutropha L359PCJ were cultivated in mineral media diluted by in natura (50% and 75% v v-1) and concentrated (50% and 75% v v-1) vinasses. Higher vinasse concentrations resulted in higher cellular growth rather than PHB accumulation for both bacteria. In vinasse-based treatments, Halomonas sp. HG03 had PHB content between 19.6 – 75.2% and R. eutropha L359PCJ, 48.4 – 68.5%. 50% (v v-1) of concentrated vinasse was the most attractive condition for PHB production by both bacteria. Further experiments in CSTR bioreactors used this nutritional condition and R. eutropha L359PCJ had PHB content of 66.3%, concentrations of residual cell dry weight (rCDW) = 9.4 g L-1 and PHB = 18.6 g L-1, with YX/S = 0.16 g gGLYCEROL-1, YP/S = 0.32 g gGLYCEROL-1 and 0.25 gPHB Lh-1. Halomonas sp. HG03 had PHB content of 45.7%, rCDW = 9.8 g L-1, PHB = 8.3 g L-1 and YX/S = 0.18 g gSUCROSE-1, YP/S = 0.16 g gSUCROSE-1 and 0.12 gPHB Lh-1. Finally, cost reductions of PHB production by R. eutropha L359PCJ with concentrated vinasse-based medium were evaluated in silico by using SuperPro Designer. As a partial source of glycerol and other nutrients for PHB production by R. eutropha L359PCJ, vinasse reduced overall production costs by 13%. Simulated processes that used concentrated vinasse-based media combined with improvements of PHB productivity and higher cellular densities had production costs between US$ 3.9 – 7.5/kgPHB and 2.6 – 7.3 years of payback time.

Application of an optimized electroporation procedure for replacement of the polyhydroxyalkanoate synthase I gene inNocardia corallina

1996 ◽  
Vol 42 (7) ◽  
pp. 715-719 ◽  
Author(s):  
Henry E. Valentin ◽  
Douglas Dennis
Keyword(s):  
Growth Conditions ◽  
Gene Replacement ◽  
Kanamycin Resistance ◽  
Pha Synthase ◽  
Wild Type ◽  
Kanamycin Resistance Gene ◽  
Gene Encoding ◽  
Nocardia Corallina ◽  
Polyhydroxyalkanoate Synthase ◽  
Type Gene

To develop a system for gene replacement in Nocardia corallina, a protocol for electroporation was optimized by systematic alterations of growth conditions, field strength, time constant and the electroporation buffer. Transformation efficiencies of 0.5 × 106–3 × 106transformants/μg plasmid DNA were obtained routinely. The gene encoding the polyhydroxyalkanoate (PHA) synthase I of N. corallina was cloned and interrupted by insertion of a kanamycin-resistance gene. The resulting plasmid was introduced into N. corallina by electroporation to inactivate the wild-type gene by homologous recombination. Kanamycin-resistant clones were screened by Southern hybridization for the absence of the wild-type gene and analyzed for PHA accumulation.Key words: gene replacement, Nocardia, polyhydroxyalkanoates (PHAs).

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