Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

Fukui, Toshiaki; Ohsawa, Kei; Mifune, Jun; Orita, Izumi; Nakamura, Satoshi

doi:10.1007/s00253-011-3100-2

Cited by 57 publications

(58 citation statements)

References 34 publications

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“…One milliliter of this seed culture was transferred to 10 ml fresh NB medium supplemented with 0.2% L-arabinose and incubated for 24 h on a rotary shaker. Arabinose is not used as a carbon source by R. eutropha, but it induces transcription of the genes under the control of the P BAD promoter (24). The cells intermediately accumulated PHB granules during this time.…”

Section: Methodsmentioning

confidence: 99%

“…For construction of BiFC expression plasmids, the two compatible and mobilizable broad-host-range vectors pBBR1MCS-2 (22) and pCM62 (23) were used. Fusions were expressed using the arabinose-inducible P BAD promoter (24). The genes coding for the N-and C-terminal parts of eYfp (YN, amino acids 1 to 154; YC, amino acids 155 to 238) were amplified via PCR from pEYFP-C1 (Clontech) using the primers EYFP-C1_f_NdeI and YNC1_r_XhoI and primers YC-C1_f_NdeI and EYFP-C1_r_SpeI, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria

Pfeiffer

Jendrossek

2013

Appl Environ Microbiol

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Poly(3-hydroxybutyrate) (PHB) granules are organelle-like multienzyme-polymer complexes (carbonosomes) and are widespread storage compounds in prokaryotes. The interaction of three PHB granule-bound proteins (PHB synthase PhaC1, phasin PhaP5, and PHB/DNA binding protein PhaM) was studied in vivo by bimolecular fluorescence complementation (BiFC) microscopy in Ralstonia eutropha. To this end, a mobilizable 2-plasmid system for arabinose-controlled expression of protein fusions with the N-terminal (YN) and C-terminal (YC) parts of the enhanced yellow fluorescent protein (eYfp) in Gram-negative bacteria was developed. Both plasmids were stably expressed in Escherichia coli and in transconjugants of R. eutropha. Homooligomerization of PhaC1, PhaP5, and PhaM and interactions between PhaC1 and PhaM and between PhaM and PhaP5 were detected in R. eutropha and colocalized with PHB granules under PHB-permissive conditions. PhaM-PhaC1 complexes were detected near the midcell/nucleoid region in the absence of PHB. Expression of BiFC complexes in R. eutropha with PhaM (PhaM homo-oligomers or PhaM-PhaC1 or PhaM-PhaP5 complexes) resulted in substantial cell elongation compared to wildtype cells and in BiFC signals that were generally located near the midcell/nucleoid region. Western blot analysis of wild-type cell extracts and proteome analysis of PHB granule-bound proteins revealed that PhaM and PhaP5 are expressed in R. eutropha and that PhaM is constitutively expressed independently of the presence or absence of PHB. Size exclusion chromatography analysis in combination with cross-linking experiments of purified PhaP5-His 6 and PhaM-His 6 showed that PhaP5 forms dimers and that PhaM is present in oligomeric (dodecamer) form. Implications of this finding for subcellular PHB localization and initiation of PHB granule formation in R. eutropha will be discussed. Ralstonia eutropha is the model organism for research on microbial synthesis of short-chain-length polyhydroxyalkanoates (PHA SCL ) such as poly(3-hydroxybutyrate) (PHB) or copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHB/HV). PHB and PHB/HV are produced on an industrial scale of 10 4 to 10 5 tons per annum worldwide. For overviews on polyhydroxyalkanoate (PHA) metabolism, see references 1 to 8. Despite many years of intensive research on the molecular biology of PHA accumulation in Eubacteria and Archaea (9, 10), several aspects of PHA granule formation and subcellular localization of PHB granules have not been solved. Two models of PHB granule initiation, (i) the budding model (PHB granules initiate in the cytoplasm membrane and the growing PHB granule blebs out from the membrane at late stages) and (ii) the micelle model (PHB synthase PhaC1 is soluble in the cytoplasm and forms micelles with other PhaC1 molecules and with the growing [hydrophobic] PHB chain) were discussed in the past (11). Recently, association of PHB granules with the cytoplasm membrane could be excluded in R. eutropha by electron cryotomography (12). In our lab, we identified two new pro...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria

Pfeiffer

Jendrossek

2013

Appl Environ Microbiol

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“…The fragments of phaJ4a Re , phaJ4b Re , and phaJ4c Re were excised by digestion with NdeI and XhoI and then ligated with pColdII (cspA promoter, Amp r ; Takara Bio, Otsu, Shiga, Japan) at the corresponding sites to obtain pColdII-J4a, pColdII-J4b, and pColdII-J4c, respectively. For expression of the genes in R. eutropha, NdeI-XhoI-restricted fragments of phaJ4a Re , phaJ4b Re , and phaJ4c Re were inserted at the NdeI and SalI sites of pBPP (phaP1 Re promoter) (9), resulting in the construction of pBPP-J4a, pBPPJ4b, and pBPP-J4c, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…A series of recombinant plasmids for the introduction of additional copies of the respective phaJ4 gene into R. eutropha was constructed based on pBPP, a broad-host-range plasmid harboring a promoter region for phaP1 Re . It has been reported that pBPP can act as a moderately strong constitutive expression plasmid in R. eutropha (9). The resulting plasmids were individually transferred into R. eutropha strains H16C Ac and NSDG⌬A by transconjugation, and then the PHA production was examined by the recombinant strains (Table 5).…”

Section: Fig 4 Evaluation Of the Stereospecificity Of Phaj4amentioning

confidence: 99%

Characterization and Functional Analyses of R -Specific Enoyl Coenzyme A Hydratases in Polyhydroxyalkanoate-Producing Ralstonia eutropha

Kawashima

Cui

Mifune

et al. 2012

Appl Environ Microbiol

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A genome survey of polyhydroxyalkanoate (PHA)-producing Ralstonia eutropha H16 detected the presence of 16 orthologs of R-specific enoyl coenzyme A (enoyl-CoA) hydratase, among which three proteins shared high homologies with the enzyme specific to enoyl-CoAs of medium chain length encoded by phaJ4 from Pseudomonas aeruginosa (phaJ4 Pa ). The recombinant forms of the three proteins, termed PhaJ4a Re to PhaJ4c Re , actually showed enoyl-CoA hydratase activity with R specificity, and the catalytic efficiencies were elevated as the substrate chain length increased from C 4 to C 8 . PhaJ4a Re and PhaJ4b Re showed >10-foldhigher catalytic efficiency than PhaJ4c Re . The functions of the new PhaJ4 proteins were investigated using previously engineered R. eutropha strains as host strains; these strains are capable of synthesizing poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil. Deletion of phaJ4a Re from the chromosome resulted in significant decrease of 3HHx composition in the accumulated copolyester, whereas no change was observed with deletion of phaJ4b Re or phaJ4c Re , indicating that only PhaJ4a Re was one of the major enzymes supplying the (R)-3HHx-CoA monomer through ␤-oxidation. Introduction of phaJ4a Re or phaJ4b Re into the R. eutropha strains using a broad-host-range vector enhanced the 3HHx composition of the copolyesters, but the introduction of phaJ4c Re did not. The two genes were then inserted into the pha operon on chromosome 1 of the engineered R. eutropha by homologous recombination. These modifications enabled the biosynthesis of P(3HB-co-3HHx) composed of a larger 3HHx fraction without a negative impact on cell growth and PHA production on soybean oil, especially when phaJ4a Re or phaJ4b Re was tandemly introduced with phaJ Ac from Aeromonas caviae.

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“…Fukui et al (25) showed previously that among the various promoters tested, the tac promoter has the strongest activity in R. eutropha. Hence, the pha promoter from A. caviae (P Ac ) was replaced with the tac promoter (P tac ), yielding a new plasmid, pBBR1ЉC1ABP tac BktB.…”

Section: Resultsmentioning

confidence: 99%

Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

et al. 2015

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Recombinant Ralstonia eutropha strain PHB ؊ 4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1 Ps ) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB ؊ 4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB ؊ 4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyrylCoA) and acetyl-CoA to form the 3H4MV carbon backbone. Over the past few decades, plastic materials made from petroleum have caused environmental problems such as waste plastic pollution and excess emission of carbon dioxide by incineration. Polyhydroxyalkanoates (PHAs) are bacterial polyesters accumulated as intracellular carbon and energy storage material and can be used as biomass-derived biodegradable plastic. Plastic material made from PHAs is expected to solve such environmental problems due to ecofriendliness (1, 2). Poly[(R)-3-hydroxybutyrate] [P(3HB)] is the most basic PHA, which is synthesized by PHA synthase (PhaC) derived from various sources, such as Ralstonia eutropha, Delftia acidovorans, Bacillus species, Burkholderia species, and Synechocystis species (3-7). P(3HB) is not a flexible material due to its high crystallinity and, therefore, has limited commercial application.To date, various 3HB-based copolymers have been synthesized to improve P(3HB) properties by introducing second-monomer units such as 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HHx), and longer-acyl-chain monomers (8-12). Our group has previously shown that Ralstonia eutropha strain PHB Ϫ 4 (PHAnegative strain) expressing Pseudomonas sp. strain 61-3 PHA synthase 1 (PhaC1 Ps ) can biosynthesize a 3HB-based copolymer containing 3-hydroxy-4-methylvalerate (3H4MV) [P(3HB-co-3H4MV)] using sugars as the sole carbon source (13). The 3H4MV unit has a branched bulky si...

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Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

Cited by 57 publications

References 34 publications

Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria

Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria

Characterization and Functional Analyses of R -Specific Enoyl Coenzyme A Hydratases in Polyhydroxyalkanoate-Producing Ralstonia eutropha

Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

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