phaC Gene Research Articles

Comparison of four phaC genes from Haloferax mediterranei and their function in different PHBV copolymer biosyntheses in Haloarcula hispanica

BackgroundThe halophilic archaeon Haloferax mediterranei is able to accumulate large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high molar fraction of 3-hydroxyvalerate (3HV) from unrelated carbon sources. A Polyhydroxyalkanoate (PHA) synthase composed of two subunits, PhaCHme and PhaEHme, has been identified in this strain, and shown to account for the PHBV biosynthesis.ResultsWith the aid of the genome sequence of Hfx. mediterranei CGMCC 1.2087, three additional phaC genes (designated phaC1, phaC2, and phaC3) were identified, which encoded putative PhaCs. Like PhaCHme (54.8 kDa), PhaC1 (49.7 kDa) and PhaC3 (62.5 kDa) possessed the conserved motifs of type III PHA synthase, which was not observed in PhaC2 (40.4 kDa). Furthermore, the longer C terminus found in the other three PhaCs was also absent in PhaC2. Reverse transcription PCR (RT-PCR) revealed that, among the four genes, only phaCHme was transcribed under PHA-accumulating conditions in the wild-type strain. However, heterologous coexpression of phaEHme with each phaC gene in Haloarcula hispanica PHB-1 showed that all PhaCs, except PhaC2, could lead to PHBV accumulation with various 3HV fractions. The three kinds of copolymers were characterized using gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Their thermal properties changed with the variations in monomer composition as well as the different molecular weights (Mw), thus might meet various application requirements.ConclusionWe discover three cryptic phaC genes in Hfx. mediterranei, and demonstrate that genetic engineering of these newly identified phaC genes has biotechnological potential for PHBV production with tailor-made material properties.

Development of a gene knockout system for Ralstonia eutropha H16 based on the broad-host-range vector expressing a mobile group II intron

Ralstonia eutropha H16 is a Gram-negative lithoautotrophic bacterium and is one of the best biopolymer-producing bacteria. It can grow to high cell densities either under lithoautotrophic or under heterotrophic conditions, which makes it suitable for a number of biotechnological applications. Also, R. eutropha H16 can degrade various aromatic compounds for environmental applications. The mobile group II intron can be used for the rapid and specific disruption of various bacterial genes by insertion into any desired target genes. Here, we applied the mobile group II intron to R. eutropha H16 and developed a markerless gene knockout system for R. eutropha: RalsTron. As a demonstration of the system, the phaC1 gene encoding polyhydroxyalkanoate synthase was successfully knocked out in R. eutropha H16. Furthermore, this knockout system would be useful for knocking out genes in other bacteria as well because it is based on a broad-host-range vector and the mobile group II intron that minimally depends on the bacterial hosts.

The PhaD regulator controls the simultaneous expression of the pha genes involved in polyhydroxyalkanoate metabolism and turnover in Pseudomonas putida KT2442

The promoters of the pha gene cluster encoding the enzymes involved in the metabolism of polyhydroxyalkanoates (PHAs) in the model strain Pseudomonas putida KT2442 have been identified and compared. The pha locus is composed by five functional promoters upstream the phaC1, phaZ, phaC2, phaF and phaI genes (P(C1), P(Z), P(C2), P(F) and P(I) respectively). P(C1) and P(I) are the most active promoters of the pha cluster allowing the transcription of phaC1ZC2D and phaIF operons. All promoters with the sole exception of P(F) are carbon source-dependent. Their transcription profiles explain the simultaneous production of PHA depolymerase and synthases to maintain the metabolic balance and PHA turnover. Mutagenesis analyses demonstrated that PhaD, a TetR-like transcriptional regulator, behaves as a carbon source-dependent activator of the pha cluster. The phaD gene is mainly transcribed as part of the phaC1ZC2D transcription unit and controls its own transcription and that of phaIF operon. The ability of PhaD to bind the P(C1) and P(I) promoters was analysed by gel retardation and DNase I footprinting assays, demonstrating that PhaD interacts with a region of 25 bp at P(C1) promoter (named OPRc1) and a 29 bp region at P(I) promoter (named OPRi). These operators contain a single binding site formed by two inverted half sites of 6 bp separated by 8 bp which overlap the corresponding promoter boxes. The 3D model structure of PhaD activator predicts that the true effector might be a CoA-intermediate of fatty acid beta-oxidation.

Metabolic engineering for microbial production of polyhydroxyalkanoates consisting of high 3-hydroxyhexanoate content by recombinant Aeromonas hydrophila

Polyhydroxyalkanoate synthase gene phaCah in Aeromonas hydrophila strain 4AK4 was deleted and its function was replaced by phaC1ps cloned from Pseudomonas stutzeri strain 1317 which favors 3-hydroxyhexanoate (3HHx) and longer chain length monomers. Genes fadD and fadL encoding Escherichia coli acyl-CoA synthase and Pseudomonas putida KT2442 fatty acid transport protein, respectively, were introduced into the recombinant with new phaC1ps. Accumulation of a series of novel medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 80–94mol% 3HHx were observed. The recombinant accumulated 54% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in cell dry weight consisting of 94.5mol% 3HHx or 51% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) consisting of 82mol% 3HHx and 16mol% of 3HO during a two-step cultivation process under nitrogen limitation when grown on sodium hexanoate or sodium octanoate. The two polyesters containing high percentage of 3HHx are physically characterized. They could be used as biodegradable pressure sensitive adhesives, coatings, polymer binding agents in organic-solvent-free paints or a source for chiral R-3-hydroxyhexanoate.

Transformation of phaG and phaC Genes into Tobacco Chloroplast Genome and Genetic Analysis

Polyhydroxyalkanoates (PHAs) belong to the group of microbial polyesters, which are a class of polymers produced by various species of bacteria as an source of carbon and energy reserve. At present, attempts focus on using transgenic plants as bioreactors to produce PHAs. Both 3-hydroxyacyl-CoA-ACP-transferase and type II PHA synthase are the key enzymes for mcl-PHAs biosynthesis. Gene phaG encoding 3-hydroxyacyl-CoA-ACP-transferase was placed under the control of psbA-pro and psbA-ter of rice ( Oryza sativa L.) to construct phaG expression cassette, and gene phaC encoding type II PHA synthase was placed under the control of prrn and rbcL-ter of rice to construct phaC expression cassette, which were ligated with the screening marker gene aadA expression cassette prrn-aadA-TpsbA-ter. These recombined fragments were cloned between the plastid rbcL and accD genes of tobacco ( Nicotiana tabacum L.) for targeting to the large single copy region of chloroplast genome. Chloroplast expression vector of pTGC was constructed and transformed into tobacco chloroplast genome through particle bombardment. Six transplastomic tobacco plants were obtained by spectinomycin screening. PCR and Southern blot analysis confirmed the integration of phaG and phaC into chloroplast genome of T 0 and T 1 transgenic plants, and the T 1 transgenic plants exhibited homogenization. The expression of phaC and phaG at transcription level was detected by reverse transcription polymerase chain reaction (RT-PCR). Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants.

Analyses of binding sequences of the PhaR protein of Rhodobacter sphaeroides FJ1

The phaC, phaP, phaR, and phaZ genes are involved in the synthesis, accumulation, and degradation of poly-beta-hydroxybutyrate (PHB). These genes encode the PHB synthase, phasin, regulatory protein, and PHB depolymerase, respectively, and are located in the same locus in the genome of Rhodobacter sphaeroides FJ1, a purple nonsulfur bacterium capable of producing PHB. We have previously found that the PhaR protein binds to the promoter regions of phaP, phaR, and phaZ and represses their expression. In this study, we determined that PhaR binds to an 11-bp palindromic sequence, 5'-CTGCN(3)GCAG-3', located at nucleotides -69 to -59 and -97 to -87 relative to the translation start site of phaP. Substitution of the three spacer nucleotides with any three or four nucleotides in this sequence had no effect on PhaR binding, but all other base deletions or substitutions in this sequence abolished its ability to bind PhaR both in vitro and in vivo. These results suggest that PhaR regulates the expression of phaP in R. sphaeroides FJ1.

Characterization of polyhydroxyalkanoate and the phaC gene of Paracoccus seriniphilus E71 strain isolated from a polluted marine microbial mat

Polyhydroxyalakanote (PHA) was produced by the marine bacteria Paracoccus seriniphilus Strain E71. Three methods were used for screening PHA in this strain: (1) microscopic analysis, (2) specifically designed primers for amplify fragments of phaC gene from Gram negative bacteria, and (3) measurements using spectroscopy, calorimetry, thermogravimetry, and rheology. The polyhydroxyalkanoic acid synthase gene (phaC) sequence had 77% identity with the phaC gene of P. denitrificans PD1222 strain. Additionally, the translated sequence showed an 86% similarity with the amino acid sequence of the phaC gene N-terminal portion of the P. denitrificans PD1222 strain. Our phaC sequence was closely related to two phaC sequences that correspond to P. denitrificans; therefore, this is the first report of a sequence of phaC that codifies a poly-(3-hydroxyalkanoate) synthase class I, specifically a poly-beta-hydroxybutyrate polymerase from the marine bacteria Paracoccus seriniphilus. The polymer PHA of E71 melts at 167.86°C (T m), which corresponded to the fusion of the crystalline polymer and thermally degrades at 296.52°C, indicating that the biopolymer has good thermal stability. Rheology showed that this polymer behaves as a nonNewtonian fluid. All these characteristics suggest that the E71 strain produces a PHA that corresponds to the crystalline thermoplastic polymer PHB type.

Ecology and characterization of polyhydroxyalkanoate-producing microorganisms on and in plants

Polyhydroxyalkanoates are energy reserve polymers produced by bacteria to survive periods of starvation in natural habitats. Little is known about the ecology of polyhydroxyalkanoate-producing bacteria. To analyse the occurrence of this specific group on/in seven different plant species, a combined strategy containing culture-dependent and -independent methods was applied. Using microbial fingerprint techniques (single-strand conformation polymorphism analysis with specific primers for phaC gene encoding the key enzyme of the polyhydroxyalkanoate synthesis), a high number of bands were especially found for the rhizosphere. Furthermore, cluster analysis revealed plant species-specific communities. Isolation of bacteria, recognition of brightly refractile cytoplasmatic inclusions, lipophilic stainings and a PCR strategy targeted on the phaC gene were used as a culture-dependent strategy for the detection of polyhydroxyalkanoate-producing bacteria. Results again represent a high degree of plant specificity: the rhizosphere of sugar beet contained the highest number of positive strains. This was confirmed by quantitative PCR: the relative copy number of phaC was statistically and significantly enhanced in all rhizospheres in comparison with bulk soil. New polyhydroxyalkanoate-producing bacterial species were detected: for example, Burkholderia terricola, Lysobacter gummosus, Pseudomonas extremaustralis, Pseudomonas brassicacearum and Pseudomonas orientalis. Our results confirm the hypothesis that the rhizosphere is an interesting hidden reservoir for polyhydroxyalkanoate producers.

Sequence Analysis, Structure Prediction, and Functional Validation of phaC1/phaC2 Genes of Pseudomonas sp. LDC-25 and Its Importance in Polyhydroxyalkanoate Accumulation

Polyhydroxyalkanoates (PHAs) are attractive biomaterials in both conventional medical devices and tissue engineering. PHA synthase is responsible for catalyzing the formation of Polyhydroxyalkanoates (PHA), but its structural information is limited. Hence, this study focuses to predict 3D model for phaC1 and phaC2 genes of field-soil strain Pseudomonas sp. LDC-25 and to validate the functional properties through in vitro studies. The phaC1/phaC2 genes were amplified, cloned, and sequenced. The sequence analysis showed > 90% homology to phaC loci and presence of α/β hydrolase fold, but phaC2 loci of LDC-25 exhibits variation in the conserved residue (Ser is replaced by Ala). Threading approach demonstrated that Carboxylesterase (d1tqha) can be used as the modeling template. The predicted models showed the presence of conserved residues at 122 (G), 205 (S), and 236 (S). In vitro studies also supported that PHA accumulation ability was less in Pseudomonas sp. LDC-25 compared to other field isolate, Pseudomonas sp. LDC-5. FT-IR spectrum showed PHA specific peaks at 1735.62 cm−1. Results of this study would help to detect the functional domains of the protein in order to elucidate their structure/function characteristics with special emphasis on invariant conserved residues.

Isolation and heterologous expression of PHA synthesising genes from Bacillus thuringiensis R1

The polyhydroxyalkanoate biosynthesis gene locus from Bacillus thuringiensis R1 was isolated, cloned and analyzed at the molecular level. We found that a ∼5 kb SacI–ClaI digested fragment of genomic DNA from B. thuringiensis R1 encoding the PHA synthesising genes, conferred PHA producing ability to E. coli. The fragment was sequenced and found to be of 4787 bp with five open reading frames. Sequence alignment with closely related species of Bacillus in the existing database revealed that the ORFs correspond to phaP, phaQ, phaR, phaB and phaC genes. However, E. coli harboring phaP, phaQ, phaR, phaB and phaC locus produced very low PHA. Furthermore, complementation of the locus with phaA from Ralstonia eutropha increased the PHA production in the recombinant E. coli from 3.0% to 24% of cell dry mass. The putative promoter regions and ribosome binding sites were identified for each of the gene. Conserved domains for PHA synthase and aceto-acetyl-coA reductase were also identified. We hence conclude that the PHA operon of Bacillus thuringiensis R1 consists of phaP, phaQ, phaR, phaB, phaC and complementation of the same with phaA is accountable for its high PHA production.

Genetic organization of pha gene locus affects phaC expression, poly(hydroxyalkanoate) composition and granule morphology in Pseudomonas corrugata

The complete sequence of the pha locus responsible for the biosynthesis of poly(hydroxyalkanoates) (PHAs) in Pseudomonas corrugata 388 was determined. As with the other known pseudomonad pha gene loci, the one in P. corrugata 388 also consists of phaC1 (1,680 bps; PHA synthase 1), phaZ (858 bp; PHA depolymerase) and phaC2 (1,683 bp; PHA synthase 2) genes. A BLAST search showed that the nucleotide sequences of these genes and the amino-acid sequences of their respective gene products are homologous to those of P. corrugata CFBP5454 and P. mediterranea CFBP5447. A putative intrinsic transcription terminator consisting of a dyad symmetry (24 bp; Delta G = -41.8 kcals) that precedes a stretch of dA residues was located in the phaC1-phaZ intergenic region. P. corrugata mutant-clones XI 32-1 and XI 32-4 were constructed in which this intergenic region was replaced with a selectable kanamycin-resistance gene. These mutant clones when grown on oleic acid for 48 h showed 4.7-to 7.0-fold increases of phaC1 and phaC2 relative expression in comparison to the initial inoculants, whereas the parental strain showed only 1.2- to 1.4-fold increases. Furthermore, in comparison to parental P. corrugata with only a few large PHA inclusion bodies, the mutants grown on oleic acid produce numerous smaller PHA granules that line the periphery of the cells. With glucose as a substrate, XI 32-1 and XI 32-4 clones produce mcl-PHA with a high content (26-31 mol%) of the mono-unsaturated 3-hydroxydodecenoate as a repeat-unit monomer. Our results show for the first time the effects of the phaC1-phaZ intergenic region on the substrate-dependent temporal expression of phaC1 and phaC2 genes, the repeat-unit composition of mcl-PHA, and the morphology of the PHA granules.

Cloning and Partial Sequencing of phac1 and phac2 Genes Encoding Poly (3-hydroxyalkanoate) Synthases from Pseudomonas aeroginusa PTCC 1310

Cloning and Partial Sequencing of phac1 and phac2 Genes Encoding Poly (3-hydroxyalkanoate) Synthases from Pseudomonas aeroginusa PTCC 1310

Production of polyhydroxyalkanoate from starch by the native isolate Bacillus cereus CFR06

A bacterial strain that produces amylase and polyhydroxyalkanoate (PHA) was isolated, identified, and classified under the Bacillus cereus group based on 16S rRNA gene sequences and specific reaction in poly-myxin egg yolk Mannitol bromothymol blue agar (PEMBA) medium and in combination with microbiological and biochemical tests. The complete ORF of phaC gene was cloned by PCR technique and nucleotide sequences were determined. Results indicated that the phaC gene had 99% homology with phaC of B. cereus (AE016877.1), 98% with B. thuringiensis (AY331151.1), and 94% with several strains of B. anthracis and B. cereus group including Bacillus sp. INT005. However, only 90% sequence homology with phaC of B. megaterium (AF109909.2) was observed. The PHA production using different fermentable sugars was tested and it was found that the CFR06 was able to accumulate 36–60% of PHA in cell dry weight (CDW). Zymogram of amylase indicated that native strain produces an extracellular enzyme of ∼80 kDa. The potency of the organism to hydrolyze starch due to the intrinsic amylase activity was considered, and starch was used as the sole carbon source for growth and PHA production. GC, FTIR, and 1H NMR analysis of the polymer indicated that the strain was a potent polyhydroxybutyrate (PHB) producer. The bacterium accumulated about 48% PHA in CDW in a starch containing medium.

Molecular characterization of Pseudomonas sp. LDC-5 involved in accumulation of poly 3-hydroxybutyrate and medium-chain-length poly 3-hydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biological polyesters, of which, Short-Chain-Length-Medium-Chain-Length (SCL-MCL) PHA copolymers are important because of their wide range of applications. The present study focused on molecular characterization of Pseudomonas sp. LDC-5 that is identified as SCL-MCL producer. Phase contrast, fluorescent and electron microscopic observation confirmed the presence of PHA granules in Pseudomonas sp. LDC-5. PCR analysis indicated the presence of expected amplicon for SCL phaC gene ( approximately 500 bp), MCL phaC1 with phaZ ( approximately 1.3), and phaC2 with phaZ ( approximately 1.5 kb). Sequence analysis of the PHA synthase gene of Pseudomonas sp. LDC-5 revealed significant differences in phaC1 and phaC2 which were further confirmed by recombinant studies. Recombinant Escherichia coli harboring the partial phaC1 gene was able to accumulate PHA, whereas E. coli with phaC2 did not accumulate PHA as verified by fold analysis, immunoblotting, Gas Chromatography (GC), Differential scanning calorimetry (DSC), and FTIR studies. The predicted theoretical three-dimensional structure revealed that PhaC1 is consistent with alpha/beta hydrolase fold. Monomer composition showed the presence of monomer ranging from C4 to C12: 1 when glucose and sodium octanoate fed as the carbon source. DSC revealed melting temperature peak at 153.12 degrees C and glass transition (T(g)) peaks at -0.37 degrees C. Thermogravimetric analysis revealed that the polymer was stable up to 276 degrees C. Fourier Transform Infrared Spectroscopy (FT-IR) spectral analysis showed the PHA specific wave number at 1,739.67 and 1,161.07 cm(-1). The potential of Pseudomonas sp. LDC-5 and its properties are discussed.

The polyhydroxyalkanoate genes of a stress resistant Antarctic Pseudomonas are situated within a genomic island

Pseudomonas sp. 14-3 is an Antarctic bacterium that shows high stress resistance in association with high polyhydroxybutyrate (PHB) production. In this paper genes involved in PHB biosynthesis ( phaRBAC) were found within a genomic island named pha-GI. Numerous mobile elements or proteins associated with them, such as an integrase, insertion sequences, a bacterial group II intron, a complete Type I protein secretion system and IncP plasmid-related proteins were detected among the 28 ORFs identified in this large genetic element (32.3 kb). The G + C distribution was not homogeneous, likely reflecting a mosaic structure that contains regions from diverse origins. pha-GI has strong similarities with genomic islands found in diverse Proteobacteria, including Burkholderiales species and Azotobacter vinelandii. The G + C content, phylogeny inference and codon usage analysis showed that the phaBAC cluster itself has a complex mosaic structure and indicated that the phaB and phaC genes were acquired by horizontal transfer, probably derived from Burkholderiales. These results describe for the first time a pha cluster located within a genomic island, and suggest that horizontal transfer of pha genes is a mechanism of adaptability to stress conditions such as those found in the extreme Antarctic environment.

A most effective method for selecting a broad range of short and medium-chain-length polyhidroxyalcanoate producing microorganisms

A molecular approach was used for selecting polyhydroxyalcanoate (PHA)-accumulating potential Gram-negative bacteria from different genera by colony polymerase chain reaction (PCR). Three degenerate primers were designed for amplifying a fragment from PHA synthase gene ( phaC ) (Class I), phaC1 and phaC2 (Class II) genes for detecting PHA-producing bacteria. Thirty-four out of 55 bacterial strains from the old collection selected using Sudan black B staining were phaC+ . PCR was used for directly selecting 35 new collection bacterial strains; these strains were phaC+ and their ability to produce PHA was confirmed by Sudan black B staining. Four specific primers were designed on genes of Class II PHA biosynthesis operon. These primers were used for evaluating 9 strains from the old phaC+ collection; 6 showed Class II PHA synthase organisation. 34 from the old and new bacterial isolation were characterised by 16S ribosomal gene (16S rDNA) gene partial sequencing. The tool proposed here can be used for better directing PHA production based on PHA biosynthesis genes and bacterial genera. Class I or II phaC genes were detected in 9 different genera and were able to infer the type of polymer produced.

A novel-designed Escherichia coli for the production of various polyhydroxyalkanoates from inexpensive substrate mixture

To efficiently produce polyhydroxyalkanoates (PHAs) from substrate mixture containing various carbon sources, Escherichia coli phosphotransferase system (PTS) mutants were constructed, in which the carbon catabolite repression in the presence of glucose was released. When cultivated in the medium containing glucose and xylose, E. coli LR1010, harboring phaC(Re) and phaAB(Re) genes from Ralstronia eutropha, could simultaneously consume glucose and xylose and accumulate short-chain-length PHA, whereas E. coli LR1120 and LR1110, harboring phaC1 gene from Pseudomonas aeruginosa, could accumulate medium-chain-length PHA. At the same time, LR1110 could simultaneously utilize the mixture of glucose and fatty acids. Semiquantitative reverse transcription polymerase chain reaction analysis indicated that disrupting phosphoenolpyruvate: sugar PTS in this strain released the repression on fad genes.

Construction of pha‐Operon‐Defined Knockout Mutants of Pseudomonas putida KT2442 and their Applications in Poly(hydroxyalkanoate) Production

Pseudomonas putida KT2442 could accumulate medium-chain-length poly(hydroxyalkanoate)s (PHA) consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate from a wide range of carbon sources. In this study, the PHA synthase pha operon (phaC1-phaZ-phaC2) was knocked out and the vgb gene encoding vitreoscilla hemoglobin protein (VHb), which could enhance oxygen uptake rate especially at low oxygen concentration, was integrated into the P. putida KT2442 genome to replace the deleted fragment. The resulting mutant P. putida KTOY01 or gene-replaced mutant KTOY02 was used as the host to study PHA synthase properties and PHA production. Different PHA polymerase (PhaC) genes, phaC(Re) from Rastonia eutropha H16, phaC(Ac) from Aeromonas cavie, and phaC2(Ps) from Pseudomonas stutzeri 1317, were expressed in the mutant strains to test the PhaC enzyme substrate specificity. The result showed P. putida KTOY01 or KTOY02 could provide not only mcl PHA monomers but also 3-hydroxybutyrate from fatty acids, which may allow the production of copolyesters poly(3HB-co-mcl 3HA). Plasmid pCJY10 containing phaC2(Ps), phbA, and phbB genes encoding PHA polymerase, beta-ketothiolase, and acetoacetyl-CoA reductase, respectively, were transformed into P. putida KTOY01 and KTOY02. Shake-flask culture showed P. putida KTOY01 or KTOY02 (pCJY10) could accumulate poly(3HB-co-mcl 3HA) from glucose. The above result showed pha operon knockout mutant of P. putida KT2442 was a very useful host of great potential not only for studying PhaC synthase, but also for microbial production of copolyesters poly(3HB-co-mcl 3HA), which is very difficult to obtain.

Characterization of biopolymer produced by Pseudomonas sp. CMG607w of marine origin

Biopolymer (polyhydroxyalkanoate, PHA) was extracted and purified from CMG607w bacterial strain isolated from sediment of Layari River outfall to the Arabian Sea. Synthesis of PHA was substrate depended in CMG607w. In the presence of sodium gluconate mcl-PHA was synthesized at the rate of 42% cell dry mass. Under highly enriched conditions, co-production of polysaccharide and blends of poly(3-hydroxybutyrate) (PHB)/PHA was observed. PCR base strategy was used to amplify PHA polymerase genes from chromosomal DNA. Conserved sequences were observed in polymerase C1 and C2. A portion of polymerase C1 and C2 genes of the pha operon was cloned and sequenced. In CMG607w PHA biosynthsis the operon has phaC1 and C2 (polymerase 1 and polymerase 2) genes. pha polymerase C1/C2 genes of CMG607w have 98% homology to Pseudomonas aeruginosa PAO1 (AE004919). Gene sequences were submitted to GenBank under accession numbers EF028075, AY596788 and AY596790.

Inactivation of type I polyhydroxyalkanoate synthase in Aeromonas hydrophila resulted in discovery of another potential PHA synthase

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase (PhaC) produced only PHBHHx from lauric acid but not from glucose. Medium-chain-length (mcl) PHA was produced from lauric acid or glucose only when PhaC of A. hydrophila was inactivated, indicating the existence of another PHA synthase in the wild type. Using PCR cloning strategy, the potential PHA synthase gene (phaC (mcl)) was obtained from genomic DNA of the wild type and exhibited strong homology to type II PHA synthase genes of Pseudomonas strains. The phaC (mcl) gene was PCR subcloned into plasmid pBBR1MCS2 and expressed in a PHA-negative mutant of Pseudomonas putida. Recombinant P. putida synthesized mcl PHA from gluconate or octanoate. This result proved that wild type A. hydrophila possessed another type II PHA synthase, which was responsible for the synthesis of mcl PHA, besides type I PHA synthase.