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Alexander Steinbuchel - One of the best experts on this subject based on the ideXlab platform.

  • role of fatty Acid de novo biosynthesis in Polyhydroxyalkanoic Acid pha and rhamnolipid synthesis by pseudomonads establishment of the transacylase phag mediated pathway for pha biosynthesis in escherichia coli
    Applied and Environmental Microbiology, 2001
    Co-Authors: Bernd H A Rehm, Timothy A Mitsky, Alexander Steinbuchel
    Abstract:

    Since Pseudomonas aeruginosa is capable of biosynthesis of Polyhydroxyalkanoic Acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty Acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the β-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty Acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty Acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty Acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty Acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.

  • the pseudomonas aeruginosa phag gene product is involved in the synthesis of Polyhydroxyalkanoic Acid consisting of medium chain length constituents from non related carbon sources
    Fems Microbiology Letters, 2000
    Co-Authors: Nils Hoffmann, Alexander Steinbuchel, Bernd H A Rehm
    Abstract:

    We recently identified the phaGPp gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty Acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino Acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region (herein called phaGPa) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phaGPp-negative mutant P. putida PhaGN-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol% 3-hydroxyhexanoate. Furthermore, an isogenic phaGPa knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaGPa mutant did not show any difference in growth rate, but PHA accumulation from gluconate was decreased to about 40% of wild-type level, whereas from fatty Acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty Acid de novo synthesis towards PHAMCL synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.

  • metabolic routing towards Polyhydroxyalkanoic Acid synthesis in recombinant escherichia coli fadr inhibition of fatty Acid β oxidation by acrylic Acid
    Fems Microbiology Letters, 1998
    Co-Authors: Qingsheng Qi, Alexander Steinbuchel, Bernd H A Rehm
    Abstract:

    Heterologous expression of the phaC1 gene from Pseudomonas aeruginosa, which encodes one of the Polyhydroxyalkanoic Acid synthases, in Escherichia coli impaired in fatty Acid β-oxidation results in Polyhydroxyalkanoic Acid accumulation when cells were cultivated on fatty Acids. We evaluated the application of the fatty Acid β-oxidation inhibitor acrylic Acid as a tool to channel intermediates of β-oxidation to Polyhydroxyalkanoic Acid synthesis. Various E. coli strains affected in fatty Acid metabolism and the wild-type strain harboring plasmid pBHR71 were analyzed with respect to Polyhydroxyalkanoic Acid accumulation in the presence of acrylic Acid. The E. coli fadR mutant RS3097 revealed the strongest Polyhydroxyalkanoic Acid accumulation. The optimum inhibitory concentration of acrylic Acid was 0.24 mg ml−1 and caused efficient channeling of intermediates of β-oxidation to Polyhydroxyalkanoic Acid synthesis. Under these conditions and grown on decanoate E. coli RS3097 harboring plasmid pBHR71 revealed a Polyhydroxyalkanoic Acid accumulation contributing to about 60% of cellular dry weight.

  • a new metabolic link between fatty Acid de novosynthesis and Polyhydroxyalkanoic Acid synthesis the phag gene from pseudomonas putidakt2440 encodes a 3 hydroxyacyl acyl carrier protein coenzyme a transferase
    Journal of Biological Chemistry, 1998
    Co-Authors: Bernd H A Rehm, Niels Kruger, Alexander Steinbuchel
    Abstract:

    Abstract To investigate the metabolic link between fatty Acid de novo synthesis and Polyhydroxyalkanoic Acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino Acids with a molecular mass of 33,876 Da, and the amino Acid sequence exhibited 44% amino Acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201. S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon. Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source. No complementation of the rhlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressingphaG gene under lac promoter control, was obtained. Heterologous expression of phaG inPseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source. Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P. oleovorans-harboring plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.

  • synechocystis sp pcc6803 possesses a two component Polyhydroxyalkanoic Acid synthase similar to that of anoxygenic purple sulfur bacteria
    Archives of Microbiology, 1998
    Co-Authors: Silke Hein, Hailinh Tran, Alexander Steinbuchel
    Abstract:

    During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a Polyhydroxyalkanoic Acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB–4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction.

Sung Chul Yoon - One of the best experts on this subject based on the ideXlab platform.

  • simultaneous inhibition of rhamnolipid and Polyhydroxyalkanoic Acid synthesis and biofilm formation in pseudomonas aeruginosa by 2 bromoalkanoic Acids effect of inhibitor alkyl chain length
    PLOS ONE, 2013
    Co-Authors: Merced Gutierrez, Mun Hwan Choi, Jong Kook Rho, Baoxia Tian, Myeong Ok Kim, Youhee Cho, Sung Chul Yoon
    Abstract:

    Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and Polyhydroxyalkanoic Acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic Acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty Acids of 2-bromohexanoic Acid (2-BrHA), 2-bromooctanoic Acid (2-BrOA) and 2-bromodecanoic Acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa ΔphaG and ΔrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA (≥90% at 2 mM) > 2-BrOA > 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets.

  • metabolic relationship between Polyhydroxyalkanoic Acid and rhamnolipid synthesis in pseudomonas aeruginosa comparative 13c nmr analysis of the products in wild type and mutants
    Journal of Biotechnology, 2011
    Co-Authors: Mun Hwan Choi, Merced Gutierrez, Youhee Cho, Taesik Yoo, Sung Chul Yoon
    Abstract:

    Polyhydroxyalkanoic Acids (PHAs) and rhamnolipids considered as biotechnologically important compounds are simultaneously produced by Pseudomonas aeruginosa. Both are synthesized from common precursors, (R)-3-hydroxyfatty Acids. To find the probable metabolic relationship between their syntheses, we investigated the PHA and rhamnolipids production in four pha (phaC1, phaC2, phaZ, and phaG), four rhl (rhlA, rhlB, rhlR, and rhlI) and rpoS mutant strains of P. aeruginosa PA14 and PAO1 grown in minimal medium containing 70 mM fructose or 30 mM decanoic Acid. Higher PHA accumulation was found in the rhamnolipid-negative mutants than in the wild-type strains, suggesting that 3-hydroxyfatty Acid precursors become more available for PHA synthesis when rhamnolipids synthesis is absent. However, compared to the wild-type strains, rhamnolipids production was not enhanced in the four pha mutants of P. aeruginosa PA14 and PAO1 which indicates that rhamnolipids production in P. aeruginosa could be tightly regulated at the transcriptional level by a quorum-sensing response. The metabolic pathways for PHA and rhamnolipid synthesis from medium-chain-length fatty Acids were also investigated using octanoic-1-¹³C Acid. ¹³C NMR analysis revealed that the monomer-unit (R)-3-hydroxyoctanoate-1-¹³C being converted from the octanoic Acid substrate was effectively incorporated into PHA. In the rhamnolipid synthesis, the (R)-3-hydroxyoctanoate-1-¹³C is suggested to be firstly converted to (R)-3-hydroxydecanoate-1,3-¹³C via fatty Acid de novo biosynthesis pathway and then further processed into (R)-3-((R)-3-hydroxyalkanoyloxy)alkanoic Acids (HAAs) via RhlA. The ratio of mono- to dirhamnolipids in the product depended on the type of carbon sources. The rhlB mutant could be exploited as an efficient producer of the important biosurfactant HAAs (e.g., ~700 mg/L HAAs was obtained when grown on 60 mM octanoic Acid).

  • enhanced production of longer side chain Polyhydroxyalkanoic Acid with ω aromatic group substitution in phaz disrupted pseudomonas fluorescens bm07 mutant through unrelated carbon source cometabolism and salicylic Acid β oxidation inhibition
    Bioresource Technology, 2010
    Co-Authors: Mun Hwan Choi, Jong Kook Rho, Xu Ping Zhao, Sung Chul Yoon
    Abstract:

    Abstract The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) ω-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL ω-aromatic fatty Acid in the presence of salicylic Acid that is known as a β-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic Acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to ∼10 mol%. An advantage of salicylic Acid inhibition technique is that salicylic Acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL–PHA with improved physicochemical properties in P. fluorescens BM07.

  • isolation and characterization of a transposon mutant of pseudomonas fluorescens bm07 enhancing the production of Polyhydroxyalkanoic Acid but deficient in cold induced exobiopolymer production
    Fems Microbiology Letters, 2010
    Co-Authors: Xu Ping Zhao, Mun Hwan Choi, Sung Chul Yoon
    Abstract:

    Pseudomonas fluorescens BM07 is known to produce cold-induced exobiopolymer, which is mainly composed of water-insoluble hydrophobic polypeptides (up to 85%) and saccharides (8%), by decreasing the culture temperature down to as low as 10 °C. We screened for transposon insertion mutants of P. fluorescens BM07 that were unable to produce the exobiopolymer. Among the eight mutants that showed the deficiency of exobiopolymer and O-lipopolysaccharide, one mutant BM07-59 that had the highest polyhydroxyalkanoates (PHA) production was selected. The transposon inserted gene in BM07-59 was identified as galU. The disruption of the gene galU coded for the putative product, UDP-glucose pyrophosphorylase (GalU), resulted in 1.5-fold more accumulation of PHA compared with the wild-type strain from 70 mM fructose or galactose at 30 °C. Electrophoretic analysis of lipopolysaccharide showed that the mutant lacked the O-antigen lipopolysaccharide bands. The glycosyl composition of the lipopolysaccharide produced by the mutant strain was significantly different from that of the wild-type strain. We suggest that the deletion of galU could be a way to shift carbon flux efficiently from exobiopolymer toward PHA in P. fluorescens BM07.

  • shifting of the distribution of aromatic monomer units in Polyhydroxyalkanoic Acid to longer units by salicylic Acid in pseudomonas fluorescens bm07 grown with mixtures of fructose and 11 phenoxyundecanoic Acid
    Biotechnology and Bioengineering, 2009
    Co-Authors: Mun Hwan Choi, Jong Kook Rho, Ji Hoon Shim, Sung Chul Yoon
    Abstract:

    Medium-chain-length-Polyhydroxyalkanoic Acids (MCL-PHAs) formed in Pseudomonas spp. have a rather broad distribution of monomer-units whose precursors are supplied via beta-oxidation degradation of MCL fatty Acids fed as the carbon source and/or via PhaG enzyme catalyzing the acyl-group transfer from 3-hydroxyacyl-ACPs derived from acetyl-CoA to coenzyme A. It was found that salicylic Acid (SA), in a concentration dependent manner, suppressed the accumulation of PHA in Pseudomonas fluorescens BM07 from fructose as well as shifted the distribution of monomer-units derived from a MCL fatty Acid co-added as carbon source (e.g., 11-phenoxyundecanoic Acid (11-POU)) to longer monomer-units. Both SA and acrylic Acid were found to induce high accumulations of 3-ketohexanoic Acid in BM07 wild-type cells grown with n-hexanoic Acid as well as to inhibit the formation of acetyl-CoA from acetoacetyl-CoA by BM07 cell extract, suggesting that 3-ketoacyl-CoA thiolase is their common beta-oxidation target. The structural motif of acrylic Acid present in the molecular structure of SA may self-explain the similar actions of the two inhibitors. A comparison of monomer modulation between BM07 wild-type and DeltaphaG mutant cells grown on the mixtures of fructose and 11-POU revealed that both PhaG and beta-oxidation inhibitor may play a critical role in the synthesis of PHA with longer side-chain omega-functional substitutions.

John K Davies - One of the best experts on this subject based on the ideXlab platform.

  • identification of a 13 kda protein associated with the Polyhydroxyalkanoic Acid granules from acinetobacter spp
    Fems Microbiology Letters, 1995
    Co-Authors: Mark A Schembri, Ronald C Bayly, Alan A Woods, John K Davies
    Abstract:

    Proteins associated with poly-beta-hydroxybutyrate (PHB) granules were purified from four Acinetobacter strains isolated from modified activated sludge treatment plants. Four predominant proteins of 64 kDa, 41 kDa, 38 kDa and 13 kDa were identified. N-terminal amino Acid sequencing of the 64-kDa and 13-kDa proteins from Acinetobacter RA3849 identified these proteins as the products of the phaCAc and phaPAc (formerly designated ORF1) genes, respectively. The expression of the 13-kDa protein (referred to as GA13) is shown to be required for the accumulation of large amounts of PHB in a recombinant Escherichia coli strain.

  • research letteridentification of a 13 kda protein associated with the Polyhydroxyalkanoic Acid granules from acinetobacter spp
    Fems Microbiology Letters, 1995
    Co-Authors: Mark A Schembri, Ronald C Bayly, Alan A Woods, John K Davies
    Abstract:

    Proteins associated with poly-β-hydroxybutyrate (PHB) granules were purified from four Acinetobacter strains isolated from modified activated sludge treatment plants. Four predominant proteins of 64 kDa, 41 kDa, 38 kDa and 13 kDa were identified. N-terminal amino Acid sequencing of the 64-kDa and 13-kDa proteins from Acinetobacter RA3849 identified these proteins as the products of the phaCAc and phaPAc (formerly designated ORF1) genes, respectively. The expression of the 13-kDa protein (referred to as GA13) is shown to be required for the accumulation of large amounts of PHB in a recombinant Escherichia coli strain.

  • phosphate concentration regulates transcription of the acinetobacter Polyhydroxyalkanoic Acid biosynthetic genes
    Journal of Bacteriology, 1995
    Co-Authors: Mark A Schembri, Ronald C Bayly, John K Davies
    Abstract:

    The Polyhydroxyalkanoic Acid (PHA) biosynthetic gene locus was cloned and characterized from an Acinetobacter sp. isolated from activated sludge. Nucleotide sequence analysis identified three clustered genes, phaAAc (encoding a beta-ketothiolase), phaBAc (encoding an acetoacetyl coenzyme A reductase), and phaCAc (encoding a PHA synthase). In addition, an open reading frame (ORF1) with potential to encode a 13-kDa protein was identified within this locus. The sequence of the putative translational product of ORF1 does not show significant similarity to any sequences in the database. A plasmid containing the Acinetobacter pha locus conferred the ability to accumulate poly-beta-hydroxybutyrate on its Escherichia coli host. These genes appear to lie in an operon transcribed by two promoters upstream of phaBAc, an apparent constitutive promoter, and a second promoter induced by phosphate starvation and under pho regulon control. These as well as a number of additional potential transcription start points were identified by a combination of primer extension and promoter-chloramphenicol acetyltransferase gene fusion studies carried out in Acinetobacter or E. coli transformants.

  • cloning and analysis of the Polyhydroxyalkanoic Acid synthase gene from an acinetobacter sp evidence that the gene is both plasmid and chromosomally located
    Fems Microbiology Letters, 1994
    Co-Authors: Mark A Schembri, Ronald C Bayly, John K Davies
    Abstract:

    The Polyhydroxyalkanoic Acid (PHA) synthase gene (phaCAc) of a species of Acinetobacter isolated from an activated sludge treatment plant was cloned by heterologous complementation in a poly-β-hydroxybutyrate (PHB) negative mutant of Alcaligenes eutrophus. Nucleotide sequence analysis of phaCAc revelaed an open reading frame of 1770 bp with potential to encode a 67.7 kDa protein. The deduced amino Acid sequence displays high similarity to other PHA synthase proteins. Probing with an internal region of phaCAc revealed that the PHA sythase gene may be present in more than one copy and may occur at both plasmid and chromosomal locations in Acinetobacter spp. This is the first organisms for which evidence has been presented to suggest that a gene involved in PHA metabolism is plasmid-encoded. Purification of PHB granules from sucrose gradients identified proteins of 38 kDa, 41 kDa and 64 kDa which may have a role in PHB metabolism.

Bernd H A Rehm - One of the best experts on this subject based on the ideXlab platform.

  • role of fatty Acid de novo biosynthesis in Polyhydroxyalkanoic Acid pha and rhamnolipid synthesis by pseudomonads establishment of the transacylase phag mediated pathway for pha biosynthesis in escherichia coli
    Applied and Environmental Microbiology, 2001
    Co-Authors: Bernd H A Rehm, Timothy A Mitsky, Alexander Steinbuchel
    Abstract:

    Since Pseudomonas aeruginosa is capable of biosynthesis of Polyhydroxyalkanoic Acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty Acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the β-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty Acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty Acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty Acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty Acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.

  • the pseudomonas aeruginosa phag gene product is involved in the synthesis of Polyhydroxyalkanoic Acid consisting of medium chain length constituents from non related carbon sources
    Fems Microbiology Letters, 2000
    Co-Authors: Nils Hoffmann, Alexander Steinbuchel, Bernd H A Rehm
    Abstract:

    We recently identified the phaGPp gene encoding (R)-3-hydroxydecanoyl-ACP:CoA transacylase in Pseudomonas putida, which directly links the fatty Acid de novo biosynthesis and polyhydroxyalkanoate (PHA) biosynthesis. An open reading frame (ORF) of which the deduced amino Acid sequence shared about 57% identity with PhaG from P. putida was identified in the P. aeruginosa genome sequence. Its coding region (herein called phaGPa) was amplified by PCR and cloned into the vector pBBR1MCS-2 under lac promoter control. The resulting plasmid pBHR88 mediated PHA synthesis contributing to about 13% of cellular dry weight from non-related carbon sources in the phaGPp-negative mutant P. putida PhaGN-21. The PHA was composed of 5 mol% 3-hydroxydodecanoate, 61 mol% 3-hydroxydecanoate, 29 mol% 3-hydroxyoctanoate and 5 mol% 3-hydroxyhexanoate. Furthermore, an isogenic phaGPa knock-out mutant of P. aeruginosa was constructed by gene replacement. The phaGPa mutant did not show any difference in growth rate, but PHA accumulation from gluconate was decreased to about 40% of wild-type level, whereas from fatty Acids wild-type level PHA accumulation was obtained. These data suggested that PhaG from P. aeruginosa exhibits 3-hydroxyacyl-ACP:CoA transacylase activity and strongly enhances the metabolic flux from fatty Acid de novo synthesis towards PHAMCL synthesis. Therefore, a function could be assigned to the ORF present in the P. aeruginosa genome, and a second PhaG is now known.

  • metabolic routing towards Polyhydroxyalkanoic Acid synthesis in recombinant escherichia coli fadr inhibition of fatty Acid β oxidation by acrylic Acid
    Fems Microbiology Letters, 1998
    Co-Authors: Qingsheng Qi, Alexander Steinbuchel, Bernd H A Rehm
    Abstract:

    Heterologous expression of the phaC1 gene from Pseudomonas aeruginosa, which encodes one of the Polyhydroxyalkanoic Acid synthases, in Escherichia coli impaired in fatty Acid β-oxidation results in Polyhydroxyalkanoic Acid accumulation when cells were cultivated on fatty Acids. We evaluated the application of the fatty Acid β-oxidation inhibitor acrylic Acid as a tool to channel intermediates of β-oxidation to Polyhydroxyalkanoic Acid synthesis. Various E. coli strains affected in fatty Acid metabolism and the wild-type strain harboring plasmid pBHR71 were analyzed with respect to Polyhydroxyalkanoic Acid accumulation in the presence of acrylic Acid. The E. coli fadR mutant RS3097 revealed the strongest Polyhydroxyalkanoic Acid accumulation. The optimum inhibitory concentration of acrylic Acid was 0.24 mg ml−1 and caused efficient channeling of intermediates of β-oxidation to Polyhydroxyalkanoic Acid synthesis. Under these conditions and grown on decanoate E. coli RS3097 harboring plasmid pBHR71 revealed a Polyhydroxyalkanoic Acid accumulation contributing to about 60% of cellular dry weight.

  • a new metabolic link between fatty Acid de novosynthesis and Polyhydroxyalkanoic Acid synthesis the phag gene from pseudomonas putidakt2440 encodes a 3 hydroxyacyl acyl carrier protein coenzyme a transferase
    Journal of Biological Chemistry, 1998
    Co-Authors: Bernd H A Rehm, Niels Kruger, Alexander Steinbuchel
    Abstract:

    Abstract To investigate the metabolic link between fatty Acid de novo synthesis and Polyhydroxyalkanoic Acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino Acids with a molecular mass of 33,876 Da, and the amino Acid sequence exhibited 44% amino Acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201. S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon. Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source. No complementation of the rhlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressingphaG gene under lac promoter control, was obtained. Heterologous expression of phaG inPseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source. Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P. oleovorans-harboring plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.

  • in recombinant Escherichia coli (fadR):
    1998
    Co-Authors: Er Steinbuºchel, Bernd H A Rehm
    Abstract:

    Metabolic routing towards Polyhydroxyalkanoic Acid synthesi

Mun Hwan Choi - One of the best experts on this subject based on the ideXlab platform.

  • simultaneous inhibition of rhamnolipid and Polyhydroxyalkanoic Acid synthesis and biofilm formation in pseudomonas aeruginosa by 2 bromoalkanoic Acids effect of inhibitor alkyl chain length
    PLOS ONE, 2013
    Co-Authors: Merced Gutierrez, Mun Hwan Choi, Jong Kook Rho, Baoxia Tian, Myeong Ok Kim, Youhee Cho, Sung Chul Yoon
    Abstract:

    Pseudomonas aeruginosa, an opportunistic human pathogen is known to synthesize rhamnolipid and Polyhydroxyalkanoic Acid (PHA) of which the acyl-group precursors (e.g., (R)-3-hydroxydecanoic Acid) are provided through RhlA and PhaG enzyme, respectively, which have 57% gene sequence homology. The inhibitory effect of three 2-bromo-fatty Acids of 2-bromohexanoic Acid (2-BrHA), 2-bromooctanoic Acid (2-BrOA) and 2-bromodecanoic Acid (2-BrDA) was compared to get an insight into the biochemical nature of their probable dual inhibition against the two enzymes. The 2-bromo-compounds were found to inhibit rhamnolipid and PHA synthesis simultaneously in alkyl-chain-length dependent manner at several millimolar concentrations. The separate and dual inhibition of the RhlA and PhaG pathway by the 2-bromo-compounds in the wild-type cells was verified by investigating their inhibitory effects on the rhamnolipid and PHA synthesis in P. aeruginosa ΔphaG and ΔrhlA mutants. Unexpectedly, the order of inhibition strength was found 2-BrHA (≥90% at 2 mM) > 2-BrOA > 2-BrDA, equally for all of the rhamnolipids and PHA synthesis, swarming motility and biofilm formation. We suggest that the novel strongest inhibitor 2-BrHA could be potentially exploited to control the rhamnolipid-associated group behaviors of this pathogen as well as for its utilization as a lead compound in screening for antimicrobial agents based on new antimicrobial targets.

  • metabolic relationship between Polyhydroxyalkanoic Acid and rhamnolipid synthesis in pseudomonas aeruginosa comparative 13c nmr analysis of the products in wild type and mutants
    Journal of Biotechnology, 2011
    Co-Authors: Mun Hwan Choi, Merced Gutierrez, Youhee Cho, Taesik Yoo, Sung Chul Yoon
    Abstract:

    Polyhydroxyalkanoic Acids (PHAs) and rhamnolipids considered as biotechnologically important compounds are simultaneously produced by Pseudomonas aeruginosa. Both are synthesized from common precursors, (R)-3-hydroxyfatty Acids. To find the probable metabolic relationship between their syntheses, we investigated the PHA and rhamnolipids production in four pha (phaC1, phaC2, phaZ, and phaG), four rhl (rhlA, rhlB, rhlR, and rhlI) and rpoS mutant strains of P. aeruginosa PA14 and PAO1 grown in minimal medium containing 70 mM fructose or 30 mM decanoic Acid. Higher PHA accumulation was found in the rhamnolipid-negative mutants than in the wild-type strains, suggesting that 3-hydroxyfatty Acid precursors become more available for PHA synthesis when rhamnolipids synthesis is absent. However, compared to the wild-type strains, rhamnolipids production was not enhanced in the four pha mutants of P. aeruginosa PA14 and PAO1 which indicates that rhamnolipids production in P. aeruginosa could be tightly regulated at the transcriptional level by a quorum-sensing response. The metabolic pathways for PHA and rhamnolipid synthesis from medium-chain-length fatty Acids were also investigated using octanoic-1-¹³C Acid. ¹³C NMR analysis revealed that the monomer-unit (R)-3-hydroxyoctanoate-1-¹³C being converted from the octanoic Acid substrate was effectively incorporated into PHA. In the rhamnolipid synthesis, the (R)-3-hydroxyoctanoate-1-¹³C is suggested to be firstly converted to (R)-3-hydroxydecanoate-1,3-¹³C via fatty Acid de novo biosynthesis pathway and then further processed into (R)-3-((R)-3-hydroxyalkanoyloxy)alkanoic Acids (HAAs) via RhlA. The ratio of mono- to dirhamnolipids in the product depended on the type of carbon sources. The rhlB mutant could be exploited as an efficient producer of the important biosurfactant HAAs (e.g., ~700 mg/L HAAs was obtained when grown on 60 mM octanoic Acid).

  • enhanced production of longer side chain Polyhydroxyalkanoic Acid with ω aromatic group substitution in phaz disrupted pseudomonas fluorescens bm07 mutant through unrelated carbon source cometabolism and salicylic Acid β oxidation inhibition
    Bioresource Technology, 2010
    Co-Authors: Mun Hwan Choi, Jong Kook Rho, Xu Ping Zhao, Sung Chul Yoon
    Abstract:

    Abstract The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) ω-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL ω-aromatic fatty Acid in the presence of salicylic Acid that is known as a β-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic Acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to ∼10 mol%. An advantage of salicylic Acid inhibition technique is that salicylic Acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL–PHA with improved physicochemical properties in P. fluorescens BM07.

  • isolation and characterization of a transposon mutant of pseudomonas fluorescens bm07 enhancing the production of Polyhydroxyalkanoic Acid but deficient in cold induced exobiopolymer production
    Fems Microbiology Letters, 2010
    Co-Authors: Xu Ping Zhao, Mun Hwan Choi, Sung Chul Yoon
    Abstract:

    Pseudomonas fluorescens BM07 is known to produce cold-induced exobiopolymer, which is mainly composed of water-insoluble hydrophobic polypeptides (up to 85%) and saccharides (8%), by decreasing the culture temperature down to as low as 10 °C. We screened for transposon insertion mutants of P. fluorescens BM07 that were unable to produce the exobiopolymer. Among the eight mutants that showed the deficiency of exobiopolymer and O-lipopolysaccharide, one mutant BM07-59 that had the highest polyhydroxyalkanoates (PHA) production was selected. The transposon inserted gene in BM07-59 was identified as galU. The disruption of the gene galU coded for the putative product, UDP-glucose pyrophosphorylase (GalU), resulted in 1.5-fold more accumulation of PHA compared with the wild-type strain from 70 mM fructose or galactose at 30 °C. Electrophoretic analysis of lipopolysaccharide showed that the mutant lacked the O-antigen lipopolysaccharide bands. The glycosyl composition of the lipopolysaccharide produced by the mutant strain was significantly different from that of the wild-type strain. We suggest that the deletion of galU could be a way to shift carbon flux efficiently from exobiopolymer toward PHA in P. fluorescens BM07.

  • shifting of the distribution of aromatic monomer units in Polyhydroxyalkanoic Acid to longer units by salicylic Acid in pseudomonas fluorescens bm07 grown with mixtures of fructose and 11 phenoxyundecanoic Acid
    Biotechnology and Bioengineering, 2009
    Co-Authors: Mun Hwan Choi, Jong Kook Rho, Ji Hoon Shim, Sung Chul Yoon
    Abstract:

    Medium-chain-length-Polyhydroxyalkanoic Acids (MCL-PHAs) formed in Pseudomonas spp. have a rather broad distribution of monomer-units whose precursors are supplied via beta-oxidation degradation of MCL fatty Acids fed as the carbon source and/or via PhaG enzyme catalyzing the acyl-group transfer from 3-hydroxyacyl-ACPs derived from acetyl-CoA to coenzyme A. It was found that salicylic Acid (SA), in a concentration dependent manner, suppressed the accumulation of PHA in Pseudomonas fluorescens BM07 from fructose as well as shifted the distribution of monomer-units derived from a MCL fatty Acid co-added as carbon source (e.g., 11-phenoxyundecanoic Acid (11-POU)) to longer monomer-units. Both SA and acrylic Acid were found to induce high accumulations of 3-ketohexanoic Acid in BM07 wild-type cells grown with n-hexanoic Acid as well as to inhibit the formation of acetyl-CoA from acetoacetyl-CoA by BM07 cell extract, suggesting that 3-ketoacyl-CoA thiolase is their common beta-oxidation target. The structural motif of acrylic Acid present in the molecular structure of SA may self-explain the similar actions of the two inhibitors. A comparison of monomer modulation between BM07 wild-type and DeltaphaG mutant cells grown on the mixtures of fructose and 11-POU revealed that both PhaG and beta-oxidation inhibitor may play a critical role in the synthesis of PHA with longer side-chain omega-functional substitutions.