Polyhydroxyalkanoate (PHA) Synthases: The Key Enzymes of PHA Synthesis

Polyhydroxyalkanoic acids (PHAs) represent a rather complex class of polyesters that are synthesized by most genera of bacteria and members of the family Halobacteriaceae of the Archaea (Steinbuchel et al., 1997; Steinbuchel and Fuchtenbusch, 1998). Most of these prokaryotes synthesize poly(3-hydroxy-butyric acid), poly(3HB), and other PHAs as storage compounds and deposit these polyesters as insoluble inclusions in the cytoplasm. The number of 91 different constituents of PHAs that were recently compiled (Steinbuchel and Valentin, 1995) has meanwhile been outnumbered, and approximately 150 different hydroxyalkanoic acids are now known to occur as constituents of PHAs. These water-insoluble PHAs exhibit rather high molecular weights, thermoplastic and/or elastomeric features, and some other interesting physical and material properties. Therefore, and since they are biodegradable (Jendrossek et al., 1996), they are considered for several applications in the packaging industry, medicine, pharmacy, agriculture and food industry, or as raw materials for the synthesis of enantiomerically pure chemicals and the production of paints (Anderson and Dawes, 1990; Muller and Seebach, 1993; Hocking and Marchessault; 1994 Steinbuchel, 1996; Williams et al., 1999; van der Walle et al., 1999). Many prokaryotic and eukaryotic organisms are able to produce low-molecular weight poly(3HB) molecules that are complexed with other biomolecules and that occur at concentrations which are three to four orders of magnitude less than storage PHAs in the cells (Reusch and Sadoff, 1988). A few eukaryotic microorganisms such as for example Aureobasidium pullulans are able to synthesize the water-soluble polyester polymalic acid which is not synthesized by prokaryotes (Liu and Steinbuchel, 1996).

[1]  A. Steinbüchel,et al.  Characterization of the Polyhydroxyalkanoate Synthase Gene Locus of Rhodobacter Sphaeroides , 1993, Biotechnology Letters.

[2]  R. Doolittle,et al.  Progressive sequence alignment as a prerequisitetto correct phylogenetic trees , 2007, Journal of Molecular Evolution.

[3]  D. Solaiman PCR cloning of Pseudomonas resinovorans polyhydroxyalkanoate biosynthesis genes and expression in Escherichia coli , 2000, Biotechnology Letters.

[4]  A. Steinbüchel,et al.  Anaylsis of polyhydroxyalkanoic acid-biosynthesis genes of anoxygenic phototrophic bacteria reveals synthesis of a polyester exhibiting an unusal composition , 1993, Applied Microbiology and Biotechnology.

[5]  Yoshiharu Doi,et al.  Substrate specificities in hydrolysis of polyhydroxyalkanoates by microbial esterases , 1993, Biotechnology Letters.

[6]  A. Steinbüchel,et al.  Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases , 2004, Applied Microbiology and Biotechnology.

[7]  S. Wiese,et al.  A general method for identification of polyhydroxyalkanoic acid synthase genes from pseudomonads belonging to the rRNA homology group I , 2004, Applied Microbiology and Biotechnology.

[8]  A. Steinbüchel,et al.  Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea , 2004, Applied Microbiology and Biotechnology.

[9]  H. Schlegel,et al.  The isolation of mutants not accumulating poly-β-hydroxybutyric acid , 2004, Archiv für Mikrobiologie.

[10]  A. Steinbüchel,et al.  Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model. , 2002, Biochimica et biophysica acta.

[11]  Q. Qi,et al.  Polyhydroxybutyrate biosynthesis in Caulobacter crescentus: molecular characterization of the polyhydroxybutyrate synthase. , 2001, Microbiology.

[12]  A. Steinbüchel PHB and Other Polhydroxyalkanoic Acids , 2001 .

[13]  H. Nakamura,et al.  Analysis of mutational effects of a polyhydroxybutyrate (PHB) polymerase on bacterial PHB accumulation using an in vivo assay system. , 2001, FEMS microbiology letters.

[14]  A. Sinskey,et al.  Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism. , 2001, Biochemistry.

[15]  A. Steinbüchel,et al.  Biochemical and molecular basis of microbial synthesis of polyhydroxyalkanoates in microorganisms. , 2001, Advances in biochemical engineering/biotechnology.

[16]  A. Steinbüchel,et al.  Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages. , 2001, Microbiology.

[17]  A. Steinbüchel,et al.  Studies on polyhydroxyalkanoate (PHA) accumulation in a PHA synthase I-negative mutant of Burkholderia cepacia generated by homogenotization. , 2000, FEMS microbiology letters.

[18]  A. Steinbüchel,et al.  Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway , 2000, Applied Microbiology and Biotechnology.

[19]  A. Steinbüchel,et al.  Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme , 2000, Applied Microbiology and Biotechnology.

[20]  S. Salzberg,et al.  DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae , 2000, Nature.

[21]  A. Steinbüchel,et al.  In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa , 2000, Applied Microbiology and Biotechnology.

[22]  A. Steinbüchel,et al.  Exploitation of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid) , 2000, Applied Microbiology and Biotechnology.

[23]  A. Steinbüchel,et al.  PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi , 2000, Applied and Environmental Microbiology.

[24]  A. Steinbüchel,et al.  Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids , 2000, Applied Microbiology and Biotechnology.

[25]  T. Clemente,et al.  Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems , 2000, Applied Microbiology and Biotechnology.

[26]  A. Sinskey,et al.  Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase. , 2000, Biochemistry.

[27]  T. Fukui,et al.  Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis. , 2000, FEMS microbiology letters.

[28]  A. Steinbüchel,et al.  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. , 2000, FEMS microbiology letters.

[29]  A. Steinbüchel,et al.  A Novel Genetically Engineered Pathway for Synthesis of Poly(Hydroxyalkanoic Acids) in Escherichia coli , 2000, Applied and Environmental Microbiology.

[30]  A. Steinbüchel,et al.  Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli. , 2000, FEMS microbiology letters.

[31]  J. García,et al.  Novel Biodegradable Aromatic Plastics from a Bacterial Source , 1999, The Journal of Biological Chemistry.

[32]  A. Miller,et al.  Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (phaC) Gene fromChromobacterium violaceum , 1999, Applied and Environmental Microbiology.

[33]  R. Weusthuis,et al.  Development of environmentally friendly coatings and paints using medium-chain-length poly(3-hydroxyalkanoates) as the polymer binder. , 1999, International journal of biological macromolecules.

[34]  A. Steinbüchel,et al.  Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. , 1999, International journal of biological macromolecules.

[35]  David P. Martin,et al.  PHA applications: addressing the price performance issue: I. Tissue engineering. , 1999, International journal of biological macromolecules.

[36]  T. Yamane,et al.  Analyses of a Polyhydroxyalkanoic Acid Granule-Associated 16-Kilodalton Protein and Its Putative Regulator in the pha Locus of Paracoccus denitrificans , 1999, Journal of bacteriology.

[37]  A. Steinbüchel,et al.  Analysis of 4-Phosphopantetheinylation of Polyhydroxybutyrate Synthase from Ralstonia eutropha: Generation of β-Alanine Auxotrophic Tn5 Mutants and Cloning of thepanD Gene Region , 1999, Journal of bacteriology.

[38]  M. Cannon,et al.  Polyhydroxyalkanoate Inclusion Body-Associated Proteins and Coding Region in Bacillus megaterium , 1999, Journal of bacteriology.

[39]  A. Steinbüchel,et al.  A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds , 1999, Archives of Microbiology.

[40]  William Arbuthnot Sir Lane,et al.  PHA synthase from chromatium vinosum: cysteine 149 is involved in covalent catalysis. , 1999, Biochemistry.

[41]  T. Fukui,et al.  Co-expression of polyhydroxyalkanoate synthase and (R)-enoyl-CoA hydratase genes of Aeromonas caviae establishes copolyester biosynthesis pathway in Escherichia coli. , 1999, FEMS microbiology letters.

[42]  N. Rawlings,et al.  Identification of the active site of legumain links it to caspases, clostripain and gingipains in a new clan of cysteine endopeptidases , 1998, FEBS letters.

[43]  T. Fukui,et al.  Cloning and Molecular Analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) Biosynthesis Genes in Pseudomonas sp. Strain 61-3 , 1998, Journal of bacteriology.

[44]  S. Lee,et al.  Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli , 1998, Applied and Environmental Microbiology.

[45]  T. Sicheritz-Pontén,et al.  The genome sequence of Rickettsia prowazekii and the origin of mitochondria , 1998, Nature.

[46]  A. Steinbüchel,et al.  In vitro synthesis of poly(3-hydroxybutyric acid) by using an enzymatic coenzyme A recycling system. , 1998, FEMS microbiology letters.

[47]  Richard Hughey,et al.  Hidden Markov models for detecting remote protein homologies , 1998, Bioinform..

[48]  R. Lenz,et al.  Investigation of the function of proteins associated to polyhydroxyalkanoate inclusions in Pseudomonas putida BMO1. , 1998, Journal of biotechnology.

[49]  H. Valentin,et al.  Formation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by PHA synthase from Ralstonia eutropha. , 1998, Journal of biotechnology.

[50]  A. Steinbüchel,et al.  Bacterial and other biological systems for polyester production. , 1998, Trends in biotechnology.

[51]  A. Steinbüchel,et al.  Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid beta-oxidation by acrylic acid. , 1998, FEMS microbiology letters.

[52]  S. Encarnación,et al.  Poly-β-Hydroxybutyrate Turnover inAzorhizobium caulinodans Is Required for Growth and AffectsnifA Expression , 1998, Journal of bacteriology.

[53]  A. Steinbüchel,et al.  A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis. The PHAG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrier protein-coenzyme a transferase. , 1998, The Journal of biological chemistry.

[54]  T. Fukui,et al.  Genetic Analysis of Comamonas acidovoransPolyhydroxyalkanoate Synthase and Factors Affecting the Incorporation of 4-Hydroxybutyrate Monomer , 1998, Applied and Environmental Microbiology.

[55]  A. Steinbüchel,et al.  Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria , 1998, Archives of Microbiology.

[56]  K. Houmiel,et al.  Multiple β-Ketothiolases Mediate Poly(β-Hydroxyalkanoate) Copolymer Synthesis in Ralstonia eutropha , 1998 .

[57]  A. Steinbüchel,et al.  In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum , 1998, Applied Microbiology and Biotechnology.

[58]  T. Fukui,et al.  Expression and Characterization of (R)-Specific Enoyl Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biosynthesis by Aeromonas caviae , 1998, Journal of bacteriology.

[59]  A. Steinbüchel,et al.  Biosynthesis of polyesters in bacteria and recombinant organisms , 1998 .

[60]  A. Steinbüchel,et al.  Functional expression of the PHA synthase gene phaC1 from Pseudomonas aeruginosa in Escherichia coli results in poly(3-hydroxyalkanoate) synthesis. , 2006, FEMS microbiology letters.

[61]  A. Steinbüchel,et al.  Synthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa: comparison of PhaC1 and PhaC2. , 1997, FEMS microbiology letters.

[62]  B. Witholt,et al.  In vitro activities of granule-bound poly[(R)-3-hydroxyalkanoate]polymerase C1 of Pseudomonas oleovorans--development of an activity test for medium-chain-length-poly(3-hydroxyalkanoate) polymerases. , 1997, European journal of biochemistry.

[63]  M. Madigan,et al.  Polyhydroxyalkanoate production in Rhodobacter capsulatus: genes, mutants, expression, and physiology , 1997, Applied and environmental microbiology.

[64]  T. Fukui,et al.  Cloning and Analysis of the Poly ( 3-Hydroxybutyrate-co3-Hydroxyhexanoate ) Biosynthesis Genes of Aeromonas caviae , 1996 .

[65]  H. Abe,et al.  Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli , 1997, Applied Microbiology and Biotechnology.

[66]  N. Guex,et al.  SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling , 1997, Electrophoresis.

[67]  J. Schrag,et al.  Lipases and alpha/beta hydrolase fold. , 1997, Methods in enzymology.

[68]  A. Sinskey,et al.  PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo , 1997, Nature Biotechnology.

[69]  A. Steinbüchel,et al.  Investigation of poly ( β-L-malic acid) production by strains of Aureobasidium pullulans , 1996, Applied Microbiology and Biotechnology.

[70]  K. Biemann,et al.  Polyhydroxybutyrate Synthase: Evidence for Covalent Catalysis , 1996 .

[71]  D. Fischer,et al.  Protein fold recognition using sequence‐derived predictions , 1996, Protein science : a publication of the Protein Society.

[72]  S. Encarnación,et al.  Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate , 1996, Journal of bacteriology.

[73]  T. Yamane,et al.  Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans , 1996, Journal of bacteriology.

[74]  D. Jendrossek,et al.  Biodegradation of polyhydroxyalkanoic acids , 1996, Applied Microbiology and Biotechnology.

[75]  O. Peoples,et al.  Biodegradable plastics from plants , 1996 .

[76]  A. Steinbüchel,et al.  Electron microscopic observations on the macromolecular organization of the boundary layer of bacterial PHA inclusion bodies , 1996 .

[77]  C. Föllner,et al.  Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. , 1995, Canadian journal of microbiology.

[78]  A. Squartini,et al.  Poly-β-hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41 , 1995 .

[79]  D. Jendrossek,et al.  Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates) , 1995, Applied and environmental microbiology.

[80]  T. Gerngross,et al.  Enzyme-catalyzed synthesis of poly[(R)-(-)-3-hydroxybutyrate]: formation of macroscopic granules in vitro. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[81]  A. Steinbüchel,et al.  Diversity of bacterial polyhydroxyalkanoic acids , 1995 .

[82]  A. Steinbüchel,et al.  Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules , 1995, Journal of bacteriology.

[83]  A. Steinbüchel,et al.  Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus , 1995, Journal of bacteriology.

[84]  W. Pangborn,et al.  Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase. , 1995, Structure.

[85]  Y. Poirier,et al.  Synthesis of high-molecular-weight poly([R]-(-)-3-hydroxybutyrate) in transgenic Arabidopsis thaliana plant cells. , 1995, International journal of biological macromolecules.

[86]  A. Steinbüchel,et al.  Purification and characterization of the poly(hydroxyalkanoic acid) synthase from Chromatium vinosum and localization of the enzyme at the surface of poly(hydroxyalkanoic acid) granules. , 1994, European journal of biochemistry.

[87]  U Derewenda,et al.  Structure of a myristoyl-ACP-specific thioesterase from Vibrio harveyi. , 1994, Biochemistry.

[88]  A. Sinskey,et al.  Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity. , 1994, Biochemistry.

[89]  M. Schembri,et al.  Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: evidence that the gene is both plasmid and chromosomally located. , 1994, FEMS microbiology letters.

[90]  B. Rost,et al.  Combining evolutionary information and neural networks to predict protein secondary structure , 1994, Proteins.

[91]  A. Sinskey,et al.  Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules , 1993, Journal of bacteriology.

[92]  B. Rost,et al.  Prediction of protein secondary structure at better than 70% accuracy. , 1993, Journal of molecular biology.

[93]  D. Seebach,et al.  POLY(HYDROXYFETTSAUREESTER), EINE FUNFTE KLASSE VON PHYSIOLOGISCH BEDEUTSAMEN ORGANISCHEN BIOPOLYMEREN ? , 1993 .

[94]  Michael J. McInerney,et al.  Synthesis and function of polyhydroxyalkanoates in anaerobic syntrophic bacteria , 1992 .

[95]  Y. Doi,et al.  Synthesis and degradation of polyhydroxyalkanoates in Alcaligenes eutrophus , 1992 .

[96]  A. Steinbüchel,et al.  Cloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1. , 1992, European journal of biochemistry.

[97]  A. Steinbüchel,et al.  Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D. , 1992, European journal of biochemistry.

[98]  Joel L. Sussman,et al.  The α/β hydrolase fold , 1992 .

[99]  A. Steinbüchel,et al.  Physiology and molecular genetics of poly(β‐hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus , 1991, Molecular microbiology.

[100]  P. Terpstra,et al.  Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. Identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA. , 1991, The Journal of biological chemistry.

[101]  A. Steinbüchel,et al.  Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter , 1991, Journal of bacteriology.

[102]  A. Anderson,et al.  Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. , 1990, Microbiological reviews.

[103]  D. Ollis,et al.  Refined structure of dienelactone hydrolase at 1.8 A. , 1990, Journal of molecular biology.

[104]  A. Sinskey,et al.  Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). , 1989, The Journal of biological chemistry.

[105]  G. W. Haywood,et al.  The importance of PHB-synthase substrate specificity in polyhydroxyalkanoate synthesis by Alcaligenes eutrophus , 1989 .

[106]  A. Steinbüchel,et al.  Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli , 1988, Journal of bacteriology.

[107]  S. Slater,et al.  Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway , 1988, Journal of bacteriology.

[108]  R. Reusch,et al.  Putative structure and functions of a poly-beta-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[109]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[110]  J. Merrick,et al.  Metabolism of Poly-β-Hydroxybutyrate: Effect of Mild Alkaline Extraction on Native Poly-β-Hydroxybutyrate Granules , 1971, Journal of bacteriology.

[111]  J. Merrick,et al.  Metabolism of poly-beta-hydroxybutyrate. I. Purification, composition, and properties of native poly-beta-hydroxybutyrate granules from Bacillus megaterium. , 1968, Biochemistry.