Search PubMed⌕ Search

Biomedical subjects

Yoshiharu Doi

Publications and source records attributed to Yoshiharu Doi.

At least 37 records · Page 2Linked to original sources
PubMed2004

Biosynthesis and compositional regulation of poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] in recombinant ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes.

A new strategy for bacterial polyhydroxyalkanoate (PHA) production by recombinant Ralstonia eutropha PHB(-)4 harboring mutated PHA synthase genes (phaC(Ac)) from Aeromona caviae was investigated. The strain harboring wild-type phaC(Ac) gene produced a PHA copolymer consisting of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB-co-3HHx)] with 3.5 mol-% of 3HHx fraction from soybean oil. When the mutants of phaC(Ac) gene were applied to this production system, 3HHx fraction in copolymers was varied in the range of 0-5.1 mol-%. Thus, the regulation of PHA copolymer compositions has been achieved by the use of mutated PHA synthase genes.

3-Hydroxybutyric Acid↗
PubMed2004

Enzymatic hydrolysis of alpha- and beta-oligo(L-aspartic acid)s by poly(aspartic acid) hydrolases-1 and 2 from Sphingomonas sp. KT-1.

The enzymatic hydrolysis of alpha- and beta-oligo(L-aspartic acid)s by PAA hydrolase-1 and PAA hydrolase-2 (purified from Sphingomonas sp. KT-1) was performed to elucidate the mechanism of the microbial degradation by Sphingomonas sp. KT-1 of the thermally synthesized alpha,beta-poly(D,L-aspartic acid) (tPAA). GPC analysis of the hydrolyzed products of alpha- and beta-tetra(L-aspartic acid)s by PAA hydrolase-1 has showed that PAA hydrolase-1 is capable of hydrolyzing only the specific amide bonds between beta-aspartic acid units. The RP-HPLC analysis of the enzymatic hydrolysis of beta-oligo(L-aspartic acid)s (4 and 5 mers) by PAA hydrolase-1 has suggested that the enzymatic hydrolysis of beta-oligo(L-aspartic acid)s occurs via an endo-mode cleavage. In contrast, PAA hydrolase-2 hydrolyzed both alpha- and beta-oligo(L-aspartic acid)s via an exo-mode cleavage to yield L-aspartic acid as a final product. A kinetic study on the enzymatic hydrolysis of alpha-oligo(L-aspartic acid)s (3 to 7 mers) by PAA hydrolase-2 has indicated that Km values are almost independent of the number of monomer units in oligomers of 4 to 7 mers, while that Vmax values are markedly dependent on the chain length and show a maximum value at 5 mer.

Aspartic Acid↗
PubMed2004

Crystal structure, thermal behavior and enzymatic degradation of poly(tetramethylene adipate) solution-grown chain-folded lamellar crystals.

Solution-grown chain-folded lamellar single crystals of poly(tetramethylene adipate) (PTMA) were prepared from a dilute solution of 2-methyl-1-propanol by isothermal crystallization. PTMA crystals were hexagonal-shaped and polyethylene decoration of the crystals resulted in a "six cross-sector" surface morphology and showed that the average direction of chain folding is parallel to the crystal growth planes of [110] and [010]. Chain-folded lamellar crystals gave well-resolved electron diffraction diagrams corresponding to all the equatorial reflections of the X-ray fiber diagram obtained from stretched PTMA melt-quenched film (beta structure). The unit cell parameters of the beta structure of PTMA were determined as a = 0.503 nm, b = 0.732 nm and c (fiber axis) = 1.442 nm with an orthorhombic crystal system. The fiber repeat distance is appropriate for an all-trans backbone conformation for the straight stems. The setting angle, with respect to the a axis, is +/-46 degrees for the corner and center chains. Thermal behavior of lamellar crystals has been investigated by means of transmission electron microscopy (TEM) and atomic force microscopy (AFM). The lamellar thickness at the edges of the crystal increased after thermal treatment with taking the molecular chains into recrystallization parts; the holes then opened up at the thickening front of the crystal. The morphological changes of lamellar crystals after enzymatic degradation by Lipase type XIII from Pseudomonas sp. and water-soluble products were characterized by TEM, AFM, gel permeation chromatography, high performance liquid chromatography and fast atom bombardment mass spectrometry. The degradation progressed mainly from the edges of the lamellar crystals without decreasing the molecular weights and the lamellar thicknesses. The central portion of single crystals was often degraded by enzymatic attacks. This result combined with thermal behavior indicates that the loosely chain-packing region exists inside the single crystal, and that molecular chains in this region have higher mobility against thermal and enzymatic treatments.

Adipates↗
PubMed2004

Evolution of polyhydroxyalkanoate (PHA) production system by "enzyme evolution": successful case studies of directed evolution.

Biotechnological studies towards the biosynthesis of polyhydroxyalkanoates (PHAs) biopolyesters have extensively progressed through the development of various metabolic engineering strategies. Historically, efficient PHA production has been achieved using the fermentation technology of naturally occurring PHA-producing bacteria based on external substrate manipulation (1st generation), and subsequent reinforcement with recombinant gene technology (2nd generation). More recently, "enzyme evolution" is becoming the 3rd generation approach for PHA production. A break-through in the chemical synthesis of macromolecules with desirable properties was achieved by the development of prominent chemical catalysts via "catalyst evolution", as represented by a series of Ziegler-Natta catalysts. Thus, one can easily accept the concept that the molecular evolution of the biocatalysts (enzymes) relevant to PHA synthesis will provide us with a chance to create novel PHA materials with high performance. The first trial of an in vitro enzyme evolution in PHA biosynthesis was reported by our group in 2001. The following literature data, as well as our own experimental results devoted to this new approach, have been accumulated over a short time. This review article focuses specifically on the concept and current case studies of the application of "enzyme evolution" to PHA biosynthesis.

Acyltransferases↗
PubMed2004

An extra large insertion in the polyhydroxyalkanoate synthase from Delftia acidovorans DS-17: its deletion effects and relation to cellular proteolysis.

The polyhydroxyalkanoate (PHA) synthase (PhaC(Da)) from Delftia acidovorans DS-17 (formerly Comamonas acidovorans) has a unique large insertion consisting of 40 amino acid residues in the alpha/beta hydrolase fold region. In order to examine whether this insertion is necessary for enzyme function, we generated a mutant gene where the nucleotides encoding the insertion sequence were deleted [phaC(Da)del(342-381)]. The ability of the mutant PhaC(Da) lacking the insertion sequence to produce PHA in recombinant Escherichia coli JM109 was compared with that of wild-type PhaC(Da). The results revealed that the mutant enzyme had approximately one fourth the activity of the wild-type enzyme. However, there was no significant difference in PHA content accumulated in cells harboring either the mutant PhaC(Da) or wild-type PhaC(Da) nor were there any differences in the molecular masses of the produced polymers. Therefore, we have concluded that the characteristic insertion is not indispensable for PHA synthesis. Also, slight cellular proteolysis in E. coli was found specifically for wild-type PhaC(Da) by Western blot analysis. This result prompted us to further examine the proteolytic stability of PhaC(Da) in D. acidovorans. Consequently, it has been suggested that the insertion region of PhaC(Da) is susceptible to cellular proteolysis during accumulation of PHA.

Acyltransferases↗
PubMed2004

Production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid.

Transgenic techniques are used to enhance and improve crop production, and their application to the production of chemical resources in plants has been under investigation. To achieve this latter goal, multiple-gene transformation is required to improve or change plant metabolic pathways; when accomplished by plant nuclear transformation, however, this procedure is costly and time consuming. We succeeded in the metabolic engineering of the tobacco plant by introducing multiple genes within a bacteria-like operon into a plastid genome. A tobacco plastid was transformed with a polycistron consisting of the spectinomycin resistance gene and three bacterial genes for the biosynthesis of the biodegradable polyester, poly[(R)-3-hydroxybutyrate] (PHB), after modification of their ribosome binding sites. DNA and RNA analysis confirmed the insertion of the introduced genes into the plastid genome and their polycistronic expression. As the result, the transplastomic tobacco accumulated PHB in its leaves. The introduced genes and the PHB productivity were maternally inherited, avoiding genetic spread by pollen diffusion, and were maintained stably in the seed progeny. Despite the low PHB productivity, this report demonstrates the feasibility of transplastomic technology for metabolic engineering. This "phyto-fermentation" system can be applied to plant production of various chemical commodities and pharmaceuticals.

Base Sequence↗
PubMed2004

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.

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.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2003

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

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 ss-oxidation cycle. Previously, two phaJ genes (phaJ1(Pa) and phaJ2(Pa)) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3(Pa) and phaJ4(Pa)) in P. aeruginosa through a genomic database search. The abilities of the four PhaJ(Pa) proteins and the (R)-3-hydroxyacyl-acyl carrier protein [(R)-3HA-ACP] dehydrases, FabA(Pa) and FabZ(Pa), to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1(Pa) or PhaJ4(Pa) 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 PhaJ1(Pa) and PhaJ4(Pa) were performed using purified samples. Kinetic analysis revealed that only PhaJ4(Pa) exhibits almost constant maximum reaction rates (V(max)) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1(Pa), PhaJ4(Pa) has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria.

Alkanes↗
PubMed2003

Biosynthesis of 5-alkylresorcinol in rice: incorporation of a putative fatty acid unit in the 5-alkylresorcinol carbon chain.

A previously unspecified "starter" unit in the predicted biosynthesis pathway of 5-alkylresorcinols has now identified as a fatty acid or its equivalent, using an efficient 5-alkylresorcinol production system of etiolated rice seedlings. Feeding saturated, odd-carbon fatty acid ester substrates from C11 to C19 specifically and markedly increased the amount of the corresponding 5-alkylresorcinol homologs with even-carbon chains that are shorter by one carbon than those of the supplied fatty acids. The amount of these homologs depended on substrate concentration. Some of the homologs whose amounts increased had linear carbon chains and the dodecyl homolog was shown to be 5-n-dodecylresorcinol. Moreover, the 13C label in the dodecyl homolog that was biosynthesized from the [1-13C]tridecanoate substrate was localized on the C-5 carbon of the resorcinol ring. These results obviously show that the fatty acid unit acts as a direct precursor and forms the side-chain moiety of 5-n-alkylresorcinol via the predicted biosynthesis pathway.

Alkylation↗
PubMed2003

Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1.

A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734bp and encodes 578 amino acids with a molecular mass of 61,757Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.

Alteromonadaceae↗
PubMed2003

Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis, and in vitro recombination of the type II polyhydroxyalkanoate synthase gene.

Type II synthase (PhaC1(Ps)) for polyhydroxyalkanoate (PHA) from Pseudomonas sp. 61-3 was subjected to an in vitro evolution system including PCR-mediated mutagenesis in order to improve the function of PhaC1(Ps) in terms of its ability to produce poly(3-hydroxybutyrate) [P(3HB)] in recombinant Escherichia coli. Based on our established in vivo assay system, two positions (Ser325 and Gln481) where mutations provided remarkable increases in P(3HB) synthesis were identified. Saturation mutagenesis at these positions was carried out to explore whether there might be more beneficial sequences for P(3HB) synthesis than those identified in the point mutation library. As a result, five single mutants [S325C (T) and Q481M (K, R)] gave rise to highly enhanced P(3HB) synthesis. Drastically enhanced P(3HB) synthesis (up to 340- to 400-fold the amount of that of the wild type) was further achieved by generation of all five variants of the double mutants combining the codons for residues 325/481. It is feasible that the replacement of Ser (specific for type II synthase) by Thr (specific for type I synthase) at position 325 resulted in acquiring greater P(3HB) synthesis ability as exhibited by type I synthases. The other hot spot, 481, that positively contributes to enhanced P(3HB) synthesis is located adjacent to a His479, a residue that forms a putative catalytic diad that can be inferred by sequence alignment.

Acyltransferases↗
PubMed2003

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

Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ(Ac)) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid beta-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ(Ac) 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 PhaJ(Ac) 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 (C(8)) 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 (C(12)) was examined by using the mutated PhaJ(Ac) as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1(Ps)). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)) 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.

Aeromonas↗
PubMed2002

Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis.

The (R)-specific enoyl coenzyme A hydratase ((R)-hydratase) from Aeromonas caviae catalyzes the addition of a water molecule to trans-2-enoyl coenzyme A (CoA), with a chain-length of 4-6 carbons, to produce the corresponding (R)-3-hydroxyacyl-CoA. It forms a dimer of identical subunits with a molecular weight of about 14,000 and is involved in polyhydroxyalkanoate (PHA) biosynthesis. The crystal structure of the enzyme has been determined at 1.5-A resolution. The structure of the monomer consists of a five-stranded antiparallel beta-sheet and a central alpha-helix, folded into a so-called "hot dog" fold, with an overhanging segment. This overhang contains the conserved residues including the hydratase 2 motif residues. In dimeric form, two beta-sheets are associated to form an extended 10-stranded beta-sheet, and the overhangs obscure the putative active sites at the subunit interface. The active site is located deep within the substrate-binding tunnel, where Asp(31) and His(36) form a catalytic dyad. These residues are catalytically important as confirmed by site-directed mutagenesis and are possibly responsible for the activation of a water molecule and the protonation of a substrate molecule, respectively. Residues such as Leu(65) and Val(130) are situated at the bottom of the substrate-binding tunnel, defining the preference of the enzyme for the chain length of the substrate. These results provide target residues for protein engineering, which will enhance the significance of this enzyme in the production of novel PHA polymers. In addition, this study provides the first structural information of the (R)-hydratase family and may facilitate further functional studies for members of the family.

Aeromonas↗
PubMed2002

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.

Acyltransferases↗
PubMed2002

Direct observation of polyhydroxyalkanoate chains by atomic force microscopy.

Atomic force microscopy in the tapping mode was used to investigate aqueous acetone-treated polyhydroxyalkanoate (PHA) inclusions freshly isolated from a recombinant bacterium. The PHA is a copolymer containing about 95 mol% 3-hydroxybutyrate units while the rests are units of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate. Polymer chains extending to several micrometers in length were observed on glass cover slips upon the evaporation of the aqueous acetone. The polymer chains seem to exist in the form of fibrillar aggregates. The height of the microfibrils was about 1 nm. Upon prolonged standing at ambient conditions, the microfibrils dissociated into finer strands of about 0.5 nm in height. The results suggest that biosynthesized PHA are stored in the inclusions in an amorphous state but with minimal chain entanglement. This is possible because the PHA chains exist in the form of fibrillar aggregates that may be the product of a special biosynthesis mechanism.

Cupriavidus necator↗
PubMed2002

In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis.

In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.

Acyltransferases↗
PubMed2002

Synthesis of a novel class of polyhydroxyalkanoates in Arabidopsis peroxisomes, and their use in monitoring short-chain-length intermediates of beta-oxidation.

The poly[(R)-3-hydroxyalkanoate] (PHA) synthase gene (phaC(Ac)) of Aeromonas caviae FA440 was modified by adding a peroxisome targeting signal encoding the last 10 amino acids at the carboxyl-terminal of spinach glycolate oxidase. The modified gene was introduced into Arabidopsis thaliana plants by Agrobacterium-mediated transformation. The transgenic Arabidopsis plant expressed the introduced gene and its protein, and it accumulated PHA in its tissues. Gas chromatography-mass spectrometry analysis demonstrated the accumulation of a novel type of PHA, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate). This strongly suggests that short-chain-length (R)-3-hydroxyacyl-CoAs were generated from intermediates of peroxisomal beta-oxidation. It was revealed by using this transgenic plant that Tween-20 can activate peroxisomal beta-oxidation of short-chain-length fatty acids.

Acyltransferases↗