Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli

Polyhydroxyalkanoate (PHA) synthase (PhaC) from Wautersia eutropha was expressed in a wide range of production level in Escherichia coli XL1-Blue cells and its effects on PhaC activity, poly[(R)-3-hydroxybutyrate] [P(3HB)] production and its molecular weights were investigated. The production level of PhaC was controlled both by the amount of chemical inducer (isopropyl-β-d-thiogalactopyranoside, IPTG) added into the medium and the use of different copy number of plasmids. In a flask experiment, as PhaC production level in the cells increased, the PhaC activity also increased in the range of low PhaC concentration. However, PhaC activity did not further increase in the range of high PhaC concentration, probably due to the formation of inclusion body in the cells. The molecular weight of P(3HB) was found to decrease with increasing PhaC activity. This trend was also verified in high cell density cultivation using 10-l jar fermentor. Furthermore, we demonstrated that the use of low copy number plasmid and appropriate induction of PhaC expression were effective in achieving both high productivity and high molecular weight of P(3HB).     

Microbial synthesis and characterization of physiochemical properties of polyhydroxyalkanoates (PHAs) produced by bacteria isolated from activated sludge obtained from the municipal wastewater works in Hong Kong

The first objective of this study was the measurement of physical properties of P(3HB-co-3HV) copolymers with different (hydroxybutyrate) HB to (hydroxyvalerate) HV ratios produced by Bacillus cereus (TRY2) isolated from activated sludge. The 3HV PHBV copolymers were 0.05, 22.6, 39.2, 54.1, and 69.1 mol%, respectively. The second objective was to study possible wastewater treatment and production of PHAs at the same time by B. cereus (TRY2) and Pseudomonas spp. (TOB17) (both were isolated from activated sludge), recombinant Bacillus DH5α, and a combination of the above three bacteria. The results were satisfactory; the maximum COD and TOC of the sewage sludge reduced were 53.5% and 67.5%, respectively.     

Construction of recombinant Bacillus subtilis for production of polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1, and pBE2C1AB, containing one or two PHA synthse, genes, respectively. The two plasmids were inserted into Bacillus subtilis DB104 to generate modified strains, B. subtilis/pBE2C1 and B. subtilis/pBE2C1AB. The two recombinants strains were subjected to fermentation and showed PHA accumulation, the first reported example of mcl-PHA production in B. subtilis. Gas Chromatography analysis identified the compound produced by B. subtilis/pBE2C1 to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer whereas that produced by B. subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxyde-canoate-co-hydroxydodecanoate (HB-HD-HDD) polymer.     

Medical applications of biopolyesters polyhydroxyalkanoates

Microbial polyhydroxyalkanoates(PHAs) are a family of biopolyesters produced by many wild type and engineered bacteria.PHAs have diverse structures accompanied by flexible thermal and mechanical properties.Combined with their in vitro biodegradation,cell and tissue compatibility,PHAs have been studied for medical applications,especially medical implants applications,including heart valve tissue engineering,vascular tissue engineering,bone tissue engineering,cartilage tissue engineering,nerve conduit tissue engineering as well as esophagus tissue engineering.Most studies have been conducted in the authors’ lab in the past 20+ years.Recently,mechanism on PHA promoted tissue regeneration was revealed to relate to cell responses to PHA biodegradation products and cell-material interactions mediated by microRNA.Very importantly,PHA implants were found not to cause carcinogenesis during long-term implantation.Thus,PHAs should have a bright future in biomedical areas.     

Tyrosine 105 of Paucimonas lemoignei PHB depolymerase PhaZ7 is essential for polymer binding

The 3-dimensional structure of the Paucimonas lemoignei poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 has significant similarity to Bacillus subtilis lipase LipA but differs from the latter by the presence of an additional domain. Analysis of this lid-like domain revealed the presence of many hydrophobic amino acid residues including Tyr₁₀₅. In this study we constructed His-tag fusions of PhaZ7 for simplified purification and investigated the effect of amino acid exchange of eight tyrosine codons of the lid-like domain. Exchanges of Tyr₁₀₃, Tyr₁₇₂, Tyr₁₇₃, Tyr₂₀₃ or Tyr₂₀₄ to alanine or serine had no phenotype but muteins with substitution of Tyr₁₈₉, Tyr₁₉₀ and Tyr₁₀₅ to alanine showed a lag phase of the in vitro PHB depolymerase reaction. Replacement of Tyr₁₀₅ by glutamate further increased the lag phase. Binding assays of the purified PHB depolymerase proteins with the natural substrate, native PHB granules, revealed a significantly reduced binding ability of the Tyr₁₀₅Glu mutant compared to the wild type protein and confirmed that Tyr₁₀₅ is involved in interaction with the polymeric substrate.     

Biosynthesis and characterization of novel polyhydroxyalkanoate polymers with high elastic property by Cupriavidus necator PHB4 transformant

We attempted to synthesize novel polyhydroxyalkanoate (PHA) containing new 3-hydroxy-4-methylvalerate (3H4MV) monomer from the transformed strain of Cupriavidus necator PHB⁻4 harboring the PHA synthase gene of Chromobacterium sp. USM2 (phaC_Cs). Novel PHA containing SCL and MCL monomers were successfully synthesized from crude palm kernel oil (CPKO) and isocaproic acid. Results showed that P(3HB-co-1 mol% 3HV-co-3 mol% 3H4MV-co-18 mol% 3HHx) possessed higher thermal stability, higher elastomeric behavior at room temperature and higher ductility than the P(3HB-co-5 mol% 3HHx). The novel PHA film was found to possess an interesting rubber-like elasticity and flexibility property which has not been reported. The soil surface degradation study showed that the novel PHA film was degraded faster than the 3HB homopolymer and copolymer with 5 mol% of 3HHx.     

Real-time analysis of enzymatic surface-initiated polymerization using surface plasmon resonance (SPR)

The kinetics of enzymatic surface-initiated polymerization of PHB on gold surface has been examined by SPR and the resultant polymer layers characterized by AFM and FT-IR spectrometry. The immobilized enzyme catalyzed surface-initiated polymerization of 3HB-CoA, resulting in the formation of a polymer brush on the surface. The rate of polymer growth from the surface was monitored by SPR in real-time. Polymer growth as measured by the increase in the resonance angle showed no apparent lag phase during the polymerization reaction. SPR analysis also revealed that the thickness of the polymer film could be controlled by varying the initial enzyme density on the surface. The average thicknesses of the PHB film after polymerization reaction were 95, 45 and 15 nm for the surfaces that were treated with 0.5, 0.3 and 0.1*10(-6) M of enzyme, respectively. The binding of PHA synthase at different concentration to the mixed SAMs and subsequent polymerization.     

Molecular weight characterization of poly[(R)-3-hydroxybutyrate] synthesized by genetically engineered strains of Escherichia coli

This study investigated the relationship of growth conditions, host strains and molecular weights of poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesized by genetically engineered Escherichia coli. Various PHA synthases belonging to types I-IV enzymes were expressed in E. coli JM109 under the same experimental conditions, and the molecular weights of the polymers were characterized by gel permeation chromatography. The results demonstrate that P(3HB) polymers have varied molecular weights and polydispersities dependent on the characteristics of the individual PHA synthase employed. P(3HB) with high number-average molecular weights (M_n) [(1.5-4.0) × 10⁶] and narrow polydispersities (1.6-1.8) were synthesized by PHA synthases from Ralstonia eutropha (type I), Delftia acidovorans (type I) and Allochromatium vinosum (type III). Contrary to these, P(3HB) with relatively low M_n [(0.17-0.79) × 10⁶] and broad polydispersities (2.2-9.0) were synthesized by PHA synthases from Aeromonas caviae (type I), Pseudomonas sp. 61-3 (type II) and Bacillus sp. INT005 (type IV). Furthermore, the molecular weights of P(3HB) synthesized under various culture conditions, in various hosts of E. coli and by mutants of PHA synthase were characterized. It was found that, in addition to culture pH [Kusaka et al. Appl Microbiol Biotechnol 1997;47:140], other variances such as culture temperature, host strain and use of mutants are effective in changing polymer molecular weight.     

Biomedical Investigations of Biodegradable PHAs

This work is a review of the results of biomedical studies of polymer devices (films, fibers, microparticles, 3D implants) made from resorbable PHAs synthesized by the bacterium Wautersia (Ralstonia) eutropha B5786, using the technology developed at the Institute of Biophysics of the Siberian Branch of the Russian Academy of Sciences. Two types of PHAs – polyhydroxybutyrate (PHB) and a hydroxybutyrate/hydroxyvalerate copolymer (PHB/PHV) – have been proven to be biocompatible in vitro in cultures of fibroblasts, endothelial cells, hepatocytes, and osteoblasts, and in short- and long-duration experiments on animals. Polymer films and membranes have been found to be usable as scaffolds for functioning cells and monofilament suture fibers – for stitching muscular-fascial wounds and in abdominal surgery. Ectopic bone formation assay and experiments with the model of segmental osteotomy showed that 3D PHB and PHB/HA implants can be used for reparative osteogenesis. The paper reports beneficial results of using polymers to repair bone defects in oral surgery.     

聚羧基脂肪酸酯细菌合成的生长环境依赖性

聚羟基脂肪酸酯（Ｐｏｌｙｈｙｄｒｏｘｙａｌｋａｎｏａｔｅ,ＰＨＡ）是一类由许多细菌合成的、结构多变的能量和碳源的储藏物质,为了得到能合成新型ＰＨＡ的菌种,或得到能在便宜简单碳源上合成ＰＨＡ的菌种,以我们实验室开发的傅立叶红外（ＦＴ－ＩＲ）细胞无损检测技术和常规气相色谱（ＧＣ）法对全国各地的采集的不同样品中分离的菌种进行了筛选,往往在不同的地理环境中,筛选出的菌株所合成的ＰＨＡ的单体组成不同,有以含四个或五个碳原子的短链单体ＰＨＡ（Ｓｈｏｒｔ－ｃｈａｉｎ－ｌｅｎｇｔｈＰＨＡ,ｓｃｌＰＨＡ）为主,有的以含六个到十六个碳原子的中长链单体ＰＨＡ（Ｍｅｄｉｕｍ－ｃｈａｉｎ－ｌｅｎｇｔｈＰＨＡ,ｍｃｌ ＰＨＡ）为主,在我们以六种底物为碳源培养的３７１株形态不一的菌株中,有４０％的菌可以合成ＰＨＡ,而其中许多可以同时合成ＰＨ