Takeharu Tsuge

Environmental life cycle comparison of polyhydroxyalkanoates produced from renewable carbon resources by bacterial fermentation

Large-scale fermentative production of poly(3-hydroxybutyrate-co-5mol% 3-hydroxyhexanoate) [P(3HB-co-5mol% 3HHx)] from soybean oil as sole carbon source is simulated using a recombinant strain of Ralstonia eutropha harboring a polyhydroxyalkanoate (PHA) synthase gene from Aeromonas caviae. Its production costs, life cycle inventories (LCI) of energy consumption and carbon dioxide emissions from the cradle-to-factory gate are calculated and compared with the counterparts for microbial production of poly(3-hydroxybutyrate) [P(3HB)] from glucose as sole carbon source. In addition, the values of bio-based polymers are compared with those of petrochemical polymers. Annual production of 5000 tonnes of P(3HB-co-5mol% 3HHx) is estimated to cost from 3.5 to 4.5 US

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

/kg, depending on presumed production performances. Similar scale production of P(3HB) from glucose is estimated to cost 3.8–4.2 US

Metabolic improvements and use of inexpensive carbon sources in microbial production of polyhydroxyalkanoates

/kg. In contrast to the comparable production costs between P(3HB-co-5mol% 3HHx) and P(3HB), life cycle inventories of energy consumption and carbon dioxide emissions favor the former product over the latter, reflecting smaller inventories and higher production yields of soybean oil compared to glucose. The life cycle inventories of energy consumption and carbon dioxide emissions of bio-based polymers are markedly lower than those of typical petrochemical polymers.

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 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.

Optical Properties of ZnO Nanoparticles Capped with Polymers

This paper deals with the microbial production of polyhydroxyalkanoates (PHAs), biodegradable thermoplastics which perform excellently as a material, from inexpensive renewable carbon sources. To date, with the help of genetic engineering techniques, it has become possible to design several types of PHAs with different compositions and to enhance the productivities of PHAs. In addition, molecular breeding of PHA biosynthesis enzymes has been demonstrated to improve polymer production. Mutant PHA synthases generated by an in vitro evolution technique have allowed the enhanced production and quality alteration of PHAs. Furthermore, use of inexpensive renewable carbon sources, such as plant oils, waste materials, and carbon dioxide, would be a key for a reduction in PHA production cost.

Rearrangement of Gene Order in the phaCAB Operon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[(R)-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli

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.

Microbial Synthesis of Poly((R)-3-hydroxybutyrate-co-3-hydroxypropionate) from Unrelated Carbon Sources by Engineered Cupriavidus necator

Optical properties of ZnO nanoparticles capped with polymers were investigated. Polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) were used as capping reagents. ZnO nanoparticles were synthesized by the sol-gel method. Fluorescence and absorption spectra were measured. When we varied the timing of the addition of the polymer to the ZnO nanoparticle solution, the optical properties were drastically changed. When PEG was added to the solution before the synthesis of ZnO nanoparticles, the fluorescence intensity increased. At the same time, the total particle size increased, which indicated that PEG molecules had capped the ZnO nanoparticles. The capping led to surface passivation, which increased fluorescence intensity. However, when PEG was added to the solution after the synthesis of ZnO nanoparticles, the fluorescence and particle size did not change. When PVP was added to the solution before the synthesis of ZnO nanoparticles, aggregation of nanoparticles occurred. When PVP was added to the solution after the synthesis of ZnO nanoparticles, fluorescence and particle size increased. This improvement of optical properties is advantageous to the practical usage of ZnO nanoparticles, such as bioimaging.

Alteration of chain length substrate specificity of Aeromonas caviae R-enantiomer-specific enoyl-coenzyme A hydratase through site-directed mutagenesis

ABSTRACT Ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineered Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.

Identification, biosynthesis, and characterization of polyhydroxyalkanoate copolymer consisting of 3-hydroxybutyrate and 3-hydroxy-4-methylvalerate.

Cupriavidus necator was engineered aiming to synthesize poly[(R)-3-hydroxybutyrate-co-3-hydroxypropionate] copolyester, P(3HB-co-3HP), from structurally unrelated carbon sources without addition of any precursor compounds. We modified a metabolic pathway in C. necator for generation of 3-hydroxypropionyl-CoA (3HP-CoA) by introducing malonyl-CoA reductase and the 3HP-CoA synthetase domain of trifunctional propionyl-CoA synthase; both members of the 3-hydroxypropionate cycle, a novel CO(2)-fixation pathway in the green nonsulfur bacterium Chloroflexus aurantiacus . In this recombinant strain, 3HP-CoA was expected to be provided from acetyl-CoA via malonyl-CoA, and then copolymerized by the function of polyhydroxyalkanoate synthase along with (R)-3-hydroxybutyryl-CoA synthesized from two acetyl-CoA molecules. C. necator wild-type strains H16 and JMP134 harboring the two heterologous genes actually synthesized P(3HB-co-3HP) copolyester with 0.2-2.1 mol % of 3HP fraction from fructose or alkanoic acids of even carbon numbers. Enzyme assay suggested that lower activity of 3HP-CoA synthetase than that of malonyl-CoA reductase caused the limited incorporation of 3HP unit into the copolyesters synthesized by the recombinant strains. The present study demonstrates the potential of engineering metabolic pathways for production of copolyesters having favorable characteristics from inexpensive carbon resources.

Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC subunit of PHA synthase from Bacillus cereus YB-4 in recombinant Escherichia coli.

ABSTRACT Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJAc) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJAc by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJAc revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C12) was examined by using the mutated PhaJAc as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.