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

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.

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

Journal of Bacteriology ◽

10.1128/jb.183.14.4217-4226.2001 ◽

2001 ◽

Vol 183 (14) ◽

pp. 4217-4226 ◽

Cited By ~ 76

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4897-4903.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4897-4903 ◽

Cited By ~ 79

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.

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Polyhydroxyalkanoate (PHA) Polymer Accumulation and pha Gene Expression in Phenazine (phz-) and Pyrrolnitrin (prn-) Defective Mutants of Pseudomonas chlororaphis PA23

Polymers ◽

10.3390/polym10111203 ◽

2018 ◽

Vol 10 (11) ◽

pp. 1203 ◽

Cited By ~ 5

Author(s):

Parveen Sharma ◽

Riffat Munir ◽

Jocelyn Plouffe ◽

Nidhi Shah ◽

Teresa Kievit ◽

...

Keyword(s):

Gene Expression ◽

Reducing Power ◽

Pentose Phosphate ◽

Pha Synthase ◽

Pseudomonas Chlororaphis ◽

Glucose Medium ◽

Wild Type ◽

Pha Production ◽

Phenazine Production ◽

Altered Gene

Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay’s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz- (PA23-63) and phz- prn- (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz- prn- (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz- (PA23-63), prn- (PA23-8), and phz- prn- (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz- and prn- mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN.

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CpxRA Influences Xenorhabdus nematophila Colonization Initiation and Outgrowth in Steinernema carpocapsae Nematodes through Regulation of the nil Locus

Applied and Environmental Microbiology ◽

10.1128/aem.02658-08 ◽

2009 ◽

Vol 75 (12) ◽

pp. 4007-4014 ◽

Cited By ~ 19

Author(s):

Erin E. Herbert Tran ◽

Aaron W. Andersen ◽

Heidi Goodrich-Blair

Keyword(s):

Regulatory System ◽

Negative Regulator ◽

Type I ◽

Bacterial Cells ◽

Xenorhabdus Nematophila ◽

Wild Type ◽

Host Interactions ◽

Host Interaction ◽

Two Stages ◽

Mutualistic Association

ABSTRACT The gammaproteobacterium Xenorhabdus nematophila mutualistically colonizes an intestinal region of a soil-dwelling nematode and is a blood pathogen of insects. The X. nematophila CpxRA two-component regulatory system is necessary for both of these host interactions (E. Herbert et al., Appl. Environ. Microbiol. 73:7826-7836, 2007). Mutualistic association of X. nematophila with its nematode host consists of two stages: initiation, where a small number of bacterial cells establish themselves in the colonization site, and outgrowth, where these cells grow to fill the space. In this study, we show that the Cpx system is necessary for both of these stages. X. nematophila ΔcpxR1 colonized fewer nematodes than its wild-type parent and did not achieve as high a density as did the wild type within a portion of the colonized nematodes. To test whether the ΔcpxR1 host interaction phenotypes are due to its overexpression of mrxA, encoding the type I pilin subunit protein, we assessed the colonization phenotype of a ΔcpxR1 ΔmrxA1 double mutant. This mutant displayed the same colonization defect as ΔcpxR1, indicating that CpxR negative regulation of mrxA does not play a detectable role in X. nematophila-host interactions. CpxR positively regulates expression of nilA, nilB, and nilC genes necessary for nematode colonization. Here we show that the nematode colonization defect of the ΔcpxR1 mutant is rescued by elevating nil gene expression through mutation of nilR, a negative regulator of nilA, nilB, and nilC. These data suggest that the nematode colonization defect previously observed in ΔcpxR1 is caused, at least in part, by altered regulation of nilA, nilB, and nilC.

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Bioprospecting of polyhydroxyalkanoates-producing bacteria from Indonesian marine environment

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d200521 ◽

2019 ◽

Vol 20 (5) ◽

Author(s):

WATUMESA A TAN ◽

IRA WIJAYA ◽

TRESNAWATI PURWADARIA

Keyword(s):

Phylogenetic Analysis ◽

16S Rdna ◽

Marine Environment ◽

Ralstonia Eutropha ◽

Class I ◽

Pha Synthase ◽

Uncultured Bacterium ◽

Pha Production ◽

Encoding Gene

Abstract. Tan WA, Wijaya I, Purwadaria T. 2019. Bioprospecting of polyhydroxyalkanoates-producing bacteria from Indonesian marine environment. Biodiversitas 20: 1309-1315. Polyhydroxyalkanoates (PHA) are potential alternates to conventional synthetic plastics. PHA production in bacteria involves PHA synthase gene encoded by phaC. In this study, we isolated PHA-producing bacteria from the coastline and 1 mile from the coastline of three beaches in Indonesia. Further phaC detection and characterization of PHA production were conducted. The isolates were subjected to phylogenetic analysis based on 16S rDNA. Red Nile staining on minimal agar revealed that twenty-three isolates showed orange fluorescent, which indicated that they accumulated PHA in their cells. PCR detection showed the presence of PHA synthase class I-encoding gene phaC in twelve isolates. One representative amplicon was sequenced to verify its identity, in which it shared 86% similarity with the PHA synthase class I-encoding gene from an uncultured bacterium. Interestingly, the production of PHA in isolate ST.PA.75, which was closely related to Vibrio sp., was 2.1-fold higher than that in the Ralstonia eutropha JMP134 control. Three isolates showed similarity with bacterial genera and/or species for which PHA producing phenotypes had never been described before TP.SWC.20, which was closely related to Microbacterium arborescens, as well as TP.SWC.33 and TP.SWC.85, which were similar to Psychrobacter spp. Phylogenetic analysis showed that the PHA producing isolates were clustered into three phyla: γ-Proteobacteria, Actinomycetes, and Bacilli. A majority of the isolates (75%) were related to γ-Proteobacteria. In this study, we uncovered diverse novel promising strains for use in the production of PHA as a more environmentally-friendly alternative to hydrocarbon-based plastics.

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Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha

Applied Microbiology and Biotechnology ◽

10.1007/s00253-011-3362-8 ◽

2011 ◽

Vol 92 (3) ◽

pp. 509-517 ◽

Cited By ~ 31

Author(s):

Xuerong Han ◽

Yasuharu Satoh ◽

Toshifumi Satoh ◽

Ken’ichiro Matsumoto ◽

Toyoji Kakuchi ◽

...

Keyword(s):

Enzymatic Synthesis ◽

Ralstonia Eutropha ◽

Class I ◽

Pha Synthase ◽

Wild Type

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Faculty Opinions recommendation of RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2353964.2845055 ◽

2010 ◽

Author(s):

Gilbert Cote

Keyword(s):

Wild Type ◽

In Cells

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Faculty Opinions recommendation of RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2353964.2045054 ◽

2010 ◽

Author(s):

Michael Weber ◽

Daniel G Gioeli

Keyword(s):

Wild Type ◽

In Cells

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

Polymers ◽

10.3390/polym13071084 ◽

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.

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