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Trevor C Charles - One of the best experts on this subject based on the ideXlab platform.

  • poly 3 hydroxybutyrate degradation in rhizobium sinorhizobium meliloti isolation and characterization of a gene encoding 3 hydroxybutyrate dehydrogenase
    Journal of Bacteriology, 1999
    Co-Authors: Punita Aneja, Trevor C Charles
    Abstract:

    We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.

  • poly 3 hydroxybutyrate degradation in rhizobium sinorhizobium meliloti isolation and characterization of a gene encoding 3 hydroxybutyrate dehydrogenase
    Journal of Bacteriology, 1999
    Co-Authors: Punita Aneja, Trevor C Charles
    Abstract:

    We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.

Yoshiharu Doi - One of the best experts on this subject based on the ideXlab platform.

  • Thermal degradation of poly[(R)-3-hydroxybutyrate], poly[ε-caprolactone
    2020
    Co-Authors: Yoshihiro Aoyagi, Koichi Yamashita, Yoshiharu Doi
    Abstract:

    Abstract Thermal degradations of poly[(R)-3-hydroxybutyrate] (PHB), poly[e-caprolactone] (PCL), and poly[(S)-lactide] (PLA) were investigated under both isothermal and non-isothermal conditions. In isothermal degradation experiments, the three polyesters showed quite different time-dependent profiles of weight loss and the number-average degree of polymerization, reflecting their different degradation mechanisms. Thermogravimetric analysis and pyrolysis coupled with GC/MS suggest that PHB is degraded by a random chain scission (cis-elimination), while PCL is degraded by an unzipping depolymerization from the hydroxyl end of the polymer chains. In contrast, the thermal degradation behavior of PLA was very complex because various reactions occurred concurrently.

  • Enzymatic Hydrolysis of Thioester Linkages in Bacterial Poly(3-hydroxybutyrate-co-3-mercaptopropionate)s by Poly(3-hydroxybutyrate) Depolymerase Isolated from Ralstonia pickettii T1
    Polymer Journal, 2005
    Co-Authors: Bo Zhu, Ken-ichi Kasuya, Yoshiharu Doi, Shogo Tanaka, Lidan Feng, Nariaki Ishii, Yoshio Inoue
    Abstract:

    Enzymatic Hydrolysis of Thioester Linkages in Bacterial Poly(3-hydroxybutyrate- co -3-mercaptopropionate)s by Poly(3-hydroxybutyrate) Depolymerase Isolated from Ralstonia pickettii T1

  • thermal degradation of poly r 3 hydroxybutyrate poly e caprolactone and poly s lactide
    Polymer Degradation and Stability, 2002
    Co-Authors: Yoshihiro Aoyagi, Koichi Yamashita, Yoshiharu Doi
    Abstract:

    Thermal degradations of poly[(R)-3-hydroxybutyrate] (PHB), poly[e-caprolactone] (PCL), and poly[(S)-lactide] (PLA) were investigated under both isothermal and non-isothermal conditions. In isothermal degradation experiments, the three polyesters showed quite different time-dependent profiles of weight loss and the number–average degree of polymerization, reflecting their different degradation mechanisms. Thermogravimetric analysis and pyrolysis coupled with GC/MS suggest that PHB is degraded by a random chain scission (cis-elimination), while PCL is degraded by an unzipping depolymerization from the hydroxyl end of the polymer chains. In contrast, the thermal degradation behavior of PLA was very complex because various reactions occurred concurrently. # 2002 Elsevier Science Ltd. All rights reserved.

  • cloning and molecular analysis of the poly 3 hydroxybutyrate and poly 3 hydroxybutyrate co 3 hydroxyalkanoate biosynthesis genes in pseudomonas sp strain 61 3
    Journal of Bacteriology, 1998
    Co-Authors: Hiromi Matsusaki, Sumihide Manji, Kazunori Taguchi, Mikiya Kato, Toshiaki Fukui, Yoshiharu Doi
    Abstract:

    Polyhydroxyalkanoates (PHAs) are accumulated in various bacteria as intracellular carbon and energy storage material under nutrient-limited conditions (3, 26, 30). These bacterial PHAs are expected to become attractive alternatives for petrochemically based plastics, since they are biodegradable thermoplastics. More than 90 different constituent monomer units have been found (47). The PHA-producing bacteria can be broadly divided into two groups according to the number of carbon atoms in the monomeric units of the PHAs produced (44). One group of bacteria, including Ralstonia eutropha (formerly Alcaligenes eutrophus), produces short chain length PHAs with C3 to C5 monomer units, while the other group, including Pseudomonas oleovorans, produces medium chain length PHAs with C6 to C14 monomer units (3, 43). Although the majority of bacteria accumulate either short chain length PHA or medium chain length PHA, several bacteria have been found to synthesize polyesters containing both short and medium chain length 3-hydroxyalkanoic acids (3HA). The bacteria Rhodospirillum rubrum (4), Rhodocyclus gelatinosus (27), and Rhodococcus sp. (13) produced terpolyesters consisting of 3HA units of C4, C5, and C6 from hexanoate. Aeromonas caviae produced a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) (6, 8, 38). Pseudomonas strain GP4BH1 produced PHA containing 3HB and 3-hydroxyoctanoate (3HO) from octanoate and PHA containing 3HB, 3HO, and 3-hydroxydecanoate (3HD) from gluconate (46). In this bacterium, a polymer blend was suggested to be synthesized rather than a copolymer. A recombinant strain of P. oleovorans expressing R. eutropha poly(3HB) [P(3HB)] biosynthesis genes has been shown to synthesize a blend of a P(3HB) homopolymer and a copolymer of 3HHx and 3HO units when grown on octanoate (49). Both polyesters were stored as separated granules within the cells (32). In addition, Pseudomonas fluorescens and several other Pseudomonas strains were found to produce a poly(3HB-co-3HA) [P(3HB-co-3HA)] copolymer consisting of 3HA units of C4 to C12 from 3HB and 1,3-butanediol (25). Although Thiocapsa pfennigii accumulated only a P(3HB) homopolymer from various carbon sources, a recombinant P. putida strain harboring the PHA synthesis genes of T. pfennigii produced a P(3HB-co-3HHx-co-3HO) terpolymer from octanoate (28). We have reported that Pseudomonas sp. strain 61-3 isolated from soil produces a blend of a P(3HB) homopolymer and a random copolymer [P(3HB-co-3HA)] consisting of 3HA units of C4 to C12 from sugars and alkanoic acids (1, 19, 20). In addition, two different types of polyester granules were formed in the same cell (9, 21). This suggests that Pseudomonas sp. strain 61-3 possesses two types of polyester synthases with different substrate specificities, that is, polyhydroxybutyrate (PHB) synthase and PHA synthase, specific for 3HB and 3HA units ranging from C4 to C12, respectively. In this study, we cloned and sequenced the P(3HB) biosynthesis genes, as well as the P(3HB-co-3HA) biosynthesis genes, of Pseudomonas sp. strain 61-3. The substrate specificity of each polyester synthase was evaluated by heterologous expression in PHA-negative mutants of P. putida and R. eutropha. In addition, we found that the phbRPs gene product exhibits significant similarity to the AraC/XylS family of transcriptional activators and report that it is a positive regulatory protein that controls the expression of the P(3HB) biosynthesis operon.

Yoshio Inoue - One of the best experts on this subject based on the ideXlab platform.

Guo-qiang Chen - One of the best experts on this subject based on the ideXlab platform.

  • effects of uracil on crystallization and rheological property of poly r 3 hydroxybutyrate co 4 hydroxybutyrate
    Composites Part A-applied Science and Manufacturing, 2018
    Co-Authors: Xuemei Che, Guo-qiang Chen
    Abstract:

    Abstract Uracil was investigated as a nucleating agent for bacterially synthesized copolymer of R-3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB). The effects of uracil on the crystallization kinetics, melting behavior, spherulite morphology, crystalline structure and rheological behavior of P3HB4HB were investigated by differential scanning calorimetry (DSC), polarized optical microscopy (POM), wide angle X-ray diffraction (WAXD), Fourier transform infrared (FTIR) spectroscopy and rheometer. The crystallization half-times (t1/2) of P3HB4HB decreased significantly in the presence of uracil. With addition of 1 wt% uracil, the t1/2 value of P3HB4HB melt crystallizing at 95 °C decreased to 2.37 min, about 3.5% of the neat polymer. In-situ FTIR spectra revealed the clear interactions between uracil and P3HB4HB in composites, inducing some precursory structures. Furthermore, the storage and loss modulus of nucleated P3HB4HB increased exponentially compared with neat P3HB4HB. It was proposed that uracil is an environment friendly nucleating agent and rheological modifier for the P3HB4HB.

  • Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.
    Metabolic engineering, 2018
    Co-Authors: Xuemei Che, Xiaoran Jiang, Jin-chun Chen, Haoqian Zhang, Guo-qiang Chen
    Abstract:

    Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the most promising biomaterials expected to be used in a wide range of scenarios. However, its large-scale production is still hindered by the high cost. Here we report the engineering of Halomonas bluephagenesis as a low-cost platform for non-sterile and continuous fermentative production of P(3HB-co-4HB) from glucose. Two interrelated 4-hydroxybutyrate (4HB) biosynthesis pathways were constructed to guarantee 4HB monomer supply for P(3HB-co-4HB) synthesis by working in concert with 3-hydroxybutyrate (3HB) pathway. Interestingly, only 0.17 mol% 4HB in the copolymer was obtained during shake flask studies. Pathway debugging using structurally related carbon source located the failure as insufficient 4HB accumulation. Further whole genome sequencing and comparative genomic analysis identified multiple orthologs of succinate semialdehyde dehydrogenase (gabD) that may compete with 4HB synthesis flux in H. bluephagenesis. Accordingly, combinatory gene-knockout strains were constructed and characterized, through which the molar fraction of 4HB was increased by 24-fold in shake flask studies. The best-performing strain was grown on glucose as the single carbon source for 60 h under non-sterile conditions in a 7-L bioreactor, reaching 26.3 g/L of dry cell mass containing 60.5% P(3HB-co-17.04 mol%4HB). Besides, 4HB molar fraction in the copolymer can be tuned from 13 mol% to 25 mol% by controlling the residual glucose concentration in the cultures. This is the first study to achieve the production of P(3HB-co-4HB) from only glucose using Halomonas.

  • Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis
    2018
    Co-Authors: Rui Shen, Jin Yin, Rui-juan Xiang, Zhi-yu Ning, Wu-zhe Huang, Guo-qiang Chen
    Abstract:

    Promoters for the expression of heterologous genes in Halomonas bluephagenesis are quite limited, and many heterologous promoters function abnormally in this strain. Pporin, a promoter of the strongest expressed protein porin in H. bluephagenesis, is one of the few promoters available for heterologous expression in H. bluephagenesis, yet it has a fixed transcriptional activity that cannot be tuned. A stable promoter library with a wide range of activities is urgently needed. This study reports an approach to construct a promoter library based on the Pporin core region, namely, from the −35 box to the transcription start site, a spacer and an insulator. Saturation mutagenesis was conducted inside the promoter core region to significantly increase the diversity within the promoter library. The promoter library worked in both E. coli and H. bluephagenesis, covering a wide range of relative transcriptional strengths from 40 to 140 000. The library is therefore suitable for the transcription of many different heterologous genes, serving as a platform for protein expression and fine-tuned metabolic engineering of H. bluephagenesis TD01 and its derivative strains. H. bluephagenesis strains harboring the orfZ gene encoding 4HB-CoA transferase driven by selected promoters from the library were constructed, the best one produced over 100 g/L cell dry weight containing 80% poly­(3-hydroxybutyrate-co-11 mol % 4-hydroxybutyrate) with a productivity of 1.59 g/L/h after 50 h growth under nonsterile fed-batch conditions. This strain was found the best for P­(3HB-co-4HB) production in the laboratory scale

  • engineering halomonas bluephagenesis td01 for non sterile production of poly 3 hydroxybutyrate co 4 hydroxybutyrate
    Bioresource Technology, 2017
    Co-Authors: Xiangbin Chen, Haoqian Zhang, Xuemei Che, Jin Yin, Guo-qiang Chen
    Abstract:

    Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), short as P(3HB-co-4HB), was successfully produced by engineered Halomonas bluephagenesis TD01 grown in glucose and γ-butyrolactone under open non-sterile conditions. Gene orfZ encoding 4HB-CoA transferase of Clostridium kluyveri was integrated into the genome to achieve P(3HB-co-4HB) accumulation comparable to that of strains encoding orfZ on plasmids. Fed-batch cultivations conducted in 1-L and 7-L fermentors, respectively, resulted in over 70g/L cell dry weight (CDW) containing 63% P(3HB-co-12mol% 4HB) after 48h under non-sterile conditions. The processes were further scaled up in a 1000-L pilot fermentor to reach 83g/L CDW containing 61% P(3HB-co-16mol% 4HB) in 48h, with a productivity of 1.04g/L/h, again, under non-sterile conditions. The elastic P(3HB-co-16mol% 4HB) shows an elongation at break of 1022±43%. Results demonstrate that the engineered Halomonas bluephagenesis TD01 is a suitable industrial strain for large scale production under open non-sterile conditions.

  • synthesis characterization and cell compatibility of novel poly ester urethane s based on poly 3 hydroxybutyrate co 4 hydroxybutyrate and poly 3 hydroxybutyrate co 3 hydroxyhexanoate prepared by melting polymerization
    Journal of Biomaterials Science-polymer Edition, 2009
    Co-Authors: Zhifei Chen, Shaoting Cheng, Guo-qiang Chen
    Abstract:

    Novel tailor-made poly(ester urethane)s (PUs) based on microbial polyesters poly(3-hydroxybutyrate-co-4hydroxybutyrate) (P3HB4HB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) were synthesized by melting polymerization (MP) using 1,6-hexamethylene diisocyanate (HDI) as a coupling agent. A comprehensive characterization using 1H-NMR, Fourier transform infrared spectroscopy (FT-IR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), mechanical properties, static water contact angles, cell proliferation using smooth muscle cells from rabbit aorta (RaSMCs) and immortalized human keratinocytes (HaCat), and blood coagulation behavior were conducted on the synthesized PUs films. DSC showed that PU samples had a low degree of crystallinity at room temperature and became fully amorphous after a melt-quenched process. The series of tailor-made PUs based on different mass ratios of P3HB4HB and PHBHHx revealed a ductile and flexile mechanical property especially for PHBHHx-rich...

Punita Aneja - One of the best experts on this subject based on the ideXlab platform.

  • poly 3 hydroxybutyrate degradation in rhizobium sinorhizobium meliloti isolation and characterization of a gene encoding 3 hydroxybutyrate dehydrogenase
    Journal of Bacteriology, 1999
    Co-Authors: Punita Aneja, Trevor C Charles
    Abstract:

    We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.

  • poly 3 hydroxybutyrate degradation in rhizobium sinorhizobium meliloti isolation and characterization of a gene encoding 3 hydroxybutyrate dehydrogenase
    Journal of Bacteriology, 1999
    Co-Authors: Punita Aneja, Trevor C Charles
    Abstract:

    We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.