scholarly journals Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

2014 ◽  
Vol 80 (9) ◽  
pp. 2867-2873 ◽  
Author(s):  
K. Ushimaru ◽  
Y. Motoda ◽  
K. Numata ◽  
T. Tsuge
Keyword(s):  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Aeromonas Caviae

scholarly journals Engineering of Aeromonas caviae Polyhydroxyalkanoate Synthase Through Site-Directed Mutagenesis for Enhanced Polymerization of the 3-Hydroxyhexanoate Unit

2021 ◽  
Vol 9 ◽  
Author(s):  
Ken Harada ◽  
Shingo Kobayashi ◽  
Kanji Oshima ◽  
Shinichi Yoshida ◽  
Takeharu Tsuge ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Structural Information ◽  
Three Dimensional ◽  
Palm Kernel ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Pha Synthase ◽  
Directed Mutagenesis ◽  
Aeromonas Caviae ◽  
Copolymer Poly

Polyhydroxyalkanoate (PHA) synthase is an enzyme that polymerizes the acyl group of hydroxyacyl-coenzyme A (CoA) substrates. Aeromonas caviae PHA synthase (PhaCAc) is an important biocatalyst for the synthesis of a useful PHA copolymer, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. Previously, a PhaCAc mutant with double mutations in asparagine 149 (replaced by serine [N149S]) and aspartate 171 (replaced by glycine [D171G]) was generated to synthesize a 3HHx-rich P(3HB-co-3HHx) and was named PhaCAc NSDG. In this study, to further increase the 3HHx fraction in biosynthesized PHA, PhaCAc was engineered based on the three-dimensional structural information of PHA synthases. First, a homology model of PhaCAc was built to target the residues for site-directed mutagenesis. Three residues, namely tyrosine 318 (Y318), serine 389 (S389), and leucine 436 (L436), were predicted to be involved in substrate recognition by PhaCAc. These PhaCAc NSDG residues were replaced with other amino acids, and the resulting triple mutants were expressed in the engineered strain of Ralstonia eutropha for application in PHA biosynthesis from palm kernel oil. The S389T mutation allowed the synthesis of P(3HB-co-3HHx) with an increased 3HHx fraction without a significant reduction in PHA yield. Thus, a new workhorse enzyme was successfully engineered for the biosynthesis of a higher 3HHx-fraction polymer.

Cloning of the Nocardia corallina polyhydroxyalkanoate synthase gene and production of poly-(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate)

1998 ◽  
Vol 44 (7) ◽  
pp. 687-691 ◽  
Author(s):  
Brian Hall ◽  
Jennifer Baldwin ◽  
Ho Gun Rhie ◽  
Douglas Dennis
Keyword(s):  
Fatty Acids ◽  
Ralstonia Eutropha ◽  
Carbon Sources ◽  
Pha Synthase ◽  
Broad Host Range ◽  
Coding Region ◽  
Reading Frame ◽  
Nocardia Corallina ◽  
Odd Chain Fatty Acids ◽  
Chain Fatty Acids

The polyhydroxyalkanoate (PHA) synthase gene (phaCNc) from Nocardia corallina was identified in a lambda library on a 6-kb BamHI fragment. A 2.8-kb XhoII subfragment was found to contain the ntact PHA synthase. This 2.8-kb fragment was subjected to DNA sequencing and was found to contain the coding region for the PHA synthase and a small downstream open reading frame of unknown function. On the basis of DNA sequence, phaCNc is closest in homology to the PHA synthases (phaCPaI and phaCPaII) of Pseudomonas aeruginosa (approximately 41% identity and 55% similarity). The 2.8-kb XhoII fragment containing phaCNc was subcloned into broad host range mobilizable plasmids and transferred into Escherichia coli, Klebsiella aerogenes (both containing a plasmid bearing phaA and phaB from Ralstonia eutropha), and PHA-negative strains of R. eutropha and Pseudomonas putida. The recombinant strains were grown on various carbon sources and the resulting polymers were analyzed. In these strains, the PHA synthase from N. corallina was able to mediate the production of poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) containing high levels of 3-hydroxyhexanoate when grown on hexanoate and larger even-chain fatty acids and poly(3-hydroxyvalerate-co-3-hydroxyheptanoate) containing high levels of 3-hydroxyheptanoate when grown on heptanoate or larger odd-chain fatty acids. Key words: polyhydroxyalkanoates (PHAs), Nocardia corallina, biodegradable, polyester.

scholarly journals Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide

2022 ◽  
Vol 10 (1) ◽  
pp. 184
Author(s):  
Izumi Orita ◽  
Gento Unno ◽  
Risa Kato ◽  
Toshiaki Fukui
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Ralstonia Eutropha ◽  
Negative Impact ◽  
Value Added ◽  
Pha Synthase ◽  
Growth Impairment ◽  
Oxidation Pathway ◽  
Methylotrophic Growth ◽  
Value Added Products

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse b-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

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.

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 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 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 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.

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