Kazunori Taguchi

High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain

Abstract High yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha H16 and its recombinant strain PHB−4/pJRDEE32d13 (a PHA-negative mutant harboring Aeromonas caviae PHA synthase gene, phaCAc) from renewable inexpensive soybean oil was investigated. The PHA production by the wild-type strain H16 was achieved with a high dry cells weight (118–126 g/l) and a high poly[(R)-3-hydroxybutyrate] [P(3HB)] content per dry cells of 72–76% (w/w). A copolymer of 3HB with 5 mol% (R)-3-hydroxyhexanoate, P(3HB-co-5 mol% 3HHx), could be produced from soybean oil as a sole carbon source by the recombinant strain PHB−4/pJRDEE32d13 with a high dry cells weight (128–138 g/l) and a high PHA content of 71–74% (w/w). The reproducible results of PHA production in the presence of soybean oil as a sole carbon source was obtained with a high yield at a range of 0.72 to 0.76 g-PHA per g-soybean oil used.

Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid ß-oxidation

Abstract The use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria. PhaJ provides monomer units for PHA synthesis from the fatty acid s-oxidation cycle. Previously, two phaJ genes (phaJ1Pa and phaJ2Pa) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3Pa and phaJ4Pa) in P. aeruginosa through a genomic database search. The abilities of the four PhaJPa proteins and the (R)-3-hydroxyacyl-acyl carrier protein [(R)-3HA-ACP] dehydrases, FabAPa and FabZPa, to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1Pa or PhaJ4Pa in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36–41 wt.% in dry cells) consisting of mainly short- (C4–C6) and medium-chain-length (C6–C10) 3HA units, respectively. Furthermore, detailed characterizations of PhaJ1Pa and PhaJ4Pa were performed using purified samples. Kinetic analysis revealed that only PhaJ4Pa exhibits almost constant maximum reaction rates (Vmax) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1Pa, PhaJ4Pa has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbARe) and NADPH-dependent acetoacetyl-CoA reductase (PhbBRe) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB−4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1Ps gene only. In addition, recombinant strains of R. eutropha PHB−4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB−4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units.

Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalkanoate Synthase (phaC) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109

ABSTRACT Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C3 to C5, medium-chain-length (MCL) PHAs consist of monomer units of C6 to C14, and SCL-MCL PHAs consist of monomers ranging in size from C4 to C14. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C4 to C10 and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803.

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.

Improved production of poly(4-hydroxybutyrate) by Comamonas acidovorans and its freeze-fracture morphology

Production of poly(4-hydroxybutyrate) [P(4HB)] by Comamonas acidovorans JCM10181 was studied by introducing additional copies of its PHA synthase gene and the beta-ketothiolase gene. A multi-copy-number broad-host-range plasmid vector, pJRD215, was modified to contain the strong hybrid trc promoter in order to express these genes in the wild-type C. acidovorans. Increased copy-number of genes resulted in significant increase in the activities of corresponding enzymes, which could further be increased by inducing with isopropyl-beta-D-thiogalactopyranoside (IPTG), indicating that the expression is under the transcriptional control of the trc promoter. P(4HB) biosynthesis in the recombinant C. acidovorans increased 2-fold to constitute more than 60 wt% of the dry cell weight. No significant decrease in the number-average molecular weights of P(4HB) in the recombinant strain was observed when compared with that of the wild-type. Freeze-fracture electron microscopy of intracellular P(4HB) granules revealed almost similar fracture morphology to the well-known mushroom-type deformation shown by polyhydroxyalkanoates with medium-chain-length monomers.