Enhanced production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) copolymer with manipulated variables and its properties

Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M _n) of copolymers ranged from 260 × 10³ to 590 × 10³Da, and the polydispersities (M _w/M _n) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T _m), glass transition temperature (T _g), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.     

Characterization of new isolated <Emphasis Type="Italic">Ralstonia</Emphasis><Emphasis Type="Italic">eutropha</Emphasis> strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.     

Production of copolymer poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) through a one-step cultivation process

A one-step cultivation process for the production of biodegradable polymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by Cupriavidus sp. USMAA2-4 was carried out using various carbon sources. It was found that Cupriavidus sp. USMAA2-4 could produce approximately 44 wt.% copolymer of P(3HB-co-4HB) with 27 mol% 4HB composition when the combination of oleic acid and 1,4-butanediol are used as carbon sources in 60 h cultivation. The manipulation of carbon-to-nitrogen ratio (C/N) resulted in the increase of dry cell weight, PHA content as well as 4HB composition. A new strategy of introducing oleic acid and 1,4-butanediol together and separately at different concentration demonstrated different yield in PHA content ranging from 47 to 58 wt.%. The molecular weight obtained was 234 kDa (by adding 1,4-butanediol and oleic acid together) and 212 kDa (by adding 1,4-butanediol separately). The copolymer of P(3HB-co-4HB) produced by Cupriavidus sp. USMAA2-4 was detected statistically as a random copolymer when analysed by nuclear magnetic resonance (NMR) spectroscopy.     

A novel <Emphasis Type="Italic">Bacillus</Emphasis> sp. accumulating poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon substrate

The objective of this paper was to report a bacterium designated as 88D, capable of producing poly (3-hydroxybutyrate-co-3-hydroxyvalerate) [P (3HB-co-3HV)] copolymer from a single carbon source, which was isolated from a municipal sewage treatment plant in Hyderabad, India. This microorganism, based on the phenotypical features and genotypic investigations, was identified as Bacillus sp. The optimal growth of Bacillus sp. 88D occurred between 28 and 30°C and at pH 7. The strain yielded a maximum of 64.62% dry cell weight (DCW) polymer in the medium containing glucose as carbon source, which was followed by 60.46% DCW polymer in glycerol containing medium. Bacillus sp. 88D produced P (3HB-co-3HV) from glucose or glycerol, when they were used as a single carbon substrate. This bacterium produced polyhydrxybutyrate (PHB) when sodium acetate was used as sole carbon substrate. The viscosity average molecular mass (Mv) of the copolymers ranged from 523 to 627 kDa. The physical, chemical and mechanical properties of the biopolymers were characterized.     

Simple sequence repeat (SSR)-based gene diversity in <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> and <Emphasis Type="Italic">Burkholderia mallei</Emphasis>

Pathogens Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) contain a large number (> 12,000) of Simple Sequence Repeats (SSRs). To study the extent to which these features have contributed to the diversification of genes, we have conducted comparative studies with nineteen genomes of these bacteria. We found 210 genes with characteristic types of SSR variations. SSRs with nonamer repeat units were the most abundant, followed by hexamers and trimers. Amino acids with smaller and nonpolar R-groups are preferred to be encoded by the variant SSRs, perhaps due to their minimal impacts to protein functionality. A majority of these genes appears to code for surface or secreted proteins that may directly interact with the host factors during pathogenesis or other environmental factors. There also are others that encode diverse functions in the cytoplasm, and this protein variability may reflect an extensive involvement of phase variation in survival and adaptation of these pathogens.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> SP5212

Summary Production of poly(3-hydroxybutyric acid) [P(3HB)] by Rhodopseudomonas palustris SP5212 isolated in this laboratory has been optimized under phototrophic microaerophilic conditions. Cells grown in malate medium accumulated 7.7% (w/w) P(3HB) of cellular dry weight at the early stationary phase of growth. The accumulated P(3HB) however, attained 15% (w/w) of cellular dry weight when acetate (1.0%, w/v) was used as the sole carbon source under nitrogen-limiting conditions. Synthesis and accumulation of polymer was favoured by sulphate-free conditions and at a phosphate concentration sub-optimal for growth. The polymer content of cells was increased drastically (34% of cellular dry weight) when the acetate containing medium was supplemented with n-alkanoic acids. Compositional analysis by H¹ NMR revealed that these accumulated polymers were composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (3HV). The contents of 3HV in these copolymers ranged from 14 to 38 mol%.     

Enhanced neurite outgrowth of rat neural cortical cells on surface-modified films of poly(lactic-<Emphasis Type="Italic">co</Emphasis>-glycolic acid)

Neural cortical cells, isolated from prenatal rat cerebra, were grown on surface-modified poly(lactic-co-glycolic acid, 65:35) (PLGA) films coated with poly-D-lysine (PDL) with either laminin (LN), fibronectin (FN) or collagen (CN). Immunocytochemistry showed that the isolated cells were highly immunopositive for both neurofilament and MAP-2 with well-organized neurites and somatodendritic localization. The presence of PDL with LN or FN on the PLGA films was essential for increased neural cell growth. Also, PLGA films coated with either PDL/LN or PDL/FN mixtures had higher neurite outgrowth and regular differentiation.Revisions requested 30 September 2004; Revisions received 10 November 2004     

Facile synthesis of optically pure (<Emphasis Type="Italic">S</Emphasis>)-3-<Emphasis Type="Italic">p</Emphasis>-hydroxyphenyllactic acid derivatives

Summary. Optically pure (S)-3-p-hydroxyphenyllactic acid derivatives are important intermediates of peroxisome proliferator-activated receptor α/γ dual agonists and heteropeptides. Many efforts have been made for synthesis of those intermediates, but there exist some flaws yet. We observed that dielectric constants of organic solvents drastically affected diazotization of O-benzyl-L-tyrosine. Optically pure (S)-3-p-benzyloxyphenyllactic acid was obtained by simple recrystallization when DMF or DMSO of higher dielectric constant was used as a co-solvent in diazotization of O-benzyl-L-tyrosine. It was easily turned into various optically pure (S)-3-p-hydroxyphenyllactic acid derivatives.     

Genomics of the<Emphasis Type="Italic"> ccoNOQP</Emphasis>-encoded<Emphasis Type="Italic"> cbb</Emphasis><Subscript>3</Subscript> oxidase complex in bacteria

Many bacteria adapt to microoxic conditions by synthesizing a particular cytochrome c oxidase (cbb ₃) complex with a high affinity for O₂, encoded by the ccoNOQP operon. A survey of genome databases indicates that ccoNOQP sequences are widespread in all sub-branches of Proteobacteria but otherwise are found only in bacteria of the CFB group (Cytophaga, Flexibacter, Bacteroides). Our analysis of available genome sequences suggests four major strategies of regulating ccoNOQP expression in response to O₂. The most widespread strategy involves direct regulation by the O₂-responsive protein Fnr. The second strategy involves an O₂-insensitive paralogue of Fnr, FixK, whose expression is regulated by the O₂-responding FixLJ two-component system. A third strategy of mixed regulation operates in bacteria carrying both fnr and fixLJ-fixK genes. Another, not yet identified, strategy is likely to operate in the -Proteobacteria Helicobacter pylori and Campylobacter jejuni which lack fnr and fixLJ-fixK genes. The FixLJ strategy appears specific for the -subclass of Proteobacteria but is not restricted to rhizobia in which it was originally discovered.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Biosynthesis of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) copolyesters with a high molar fraction of 3-hydroxyvalerate by an insect-symbiotic <Emphasis Type="Italic">Burkholderia</Emphasis> sp. IS-01

Burkholderia sp. IS-01 capable of biosynthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] copolyesters with a high molar fraction of 3HV was isolated from the gut of the adult longicorn beetle, Moechotypa diphysis. The strain IS-01 was relatively tolerant to high concentrations of levulinic acid and accumulated a poly(13.5 mol% 3HB-co-86.5 mol% 3HV) copolyester when cultivated on a mixture of gluconate (20 g/L) and levulinic acid (12.5 g/L). In this case, the content of the copolyester in the cells was approximately 60.0%. The compositions of the copolyesters were easily regulated by altering the molar ratio of gluconate and levulinic acid in the medium. The organism was found to possess a class I PHA synthase (PhaC) gene (1,881 bp) that encodes a protein with a deduced molecular mass of 68,538 Da that consists of 626 amino acids. The PhaC of this organism was most similar to that of B. cenocepacia PC184 (92% similarity).     

Optimization of <Emphasis Type="Italic">Renealmia mexicana</Emphasis> (Klotzsch ex. Petersen) cultivation <Emphasis Type="Italic">in vitro</Emphasis>

Renealmia mexicana (Klotzsch ex. Petersen) is a tropical plant found in southern México with an ornamental value and a potential source of curcuminoids. Its distribution in Chiapas has decreased because of deforestation and low propagation and germination rate, so a protocol for in vitro propagation was developed. An orthogonal experimental design of L₉ (3⁴) in triplicate was used to investigate the effect of 6-benzyl adenine (BA), indole butyric acid (IBA), silver nitrate (AgNO₃), and sucrose on shoot, root, and leaf development of plantlets grown in vitro. Plantlets with well-developed shoots and roots were transferred to pots containing a mixture of peat moss and agrolite for hardening before transfer to soil. The Murashige and Skoog (Physiol. Plant. 15:473–497, 1962) mineral medium (MS) supplemented with 4.4 μM BA, 2.5 μM IBA, 11.7 μM AgNO_3y and 5.5% (w/v) sucrose gave most shoots, 8.9 μM BA, 2.5 μM IBA, 17.7 μM AgNO₃ and 5.5% (w/v) sucrose most roots, and 8.9 μM BA, 4.9 μM IBA, 11.7 μM AgNO₃ and 3.0% (w/v) sucrose most leaves, although other combinations were statistically equivalent in each case. Sucrose was the factor that most explained the variation in the promotion of shoots, roots, and leaves. The protocol developed resulted in up to 100% survival when plantlets were transferred to soil using AgNO₃, confirming that hardening of plantlets in vitro using hormonal stimulation was a suitable strategy to improve acclimatization.     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

Construction of genetically modified tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> cv. Petit Havana) harboring <Emphasis Type="Italic">ompH(A:3)</Emphasis> from <Emphasis Type="Italic">Pasteurella multocida</Emphasis> (A:3)

Fowl cholera, caused by Pasteurella multocida (A:3), is a fearsome disease leading to a nonproductive influence upon poultry industry. It has been known that outer membrane protein H (OmpH) in the bacterium is a strong candidate to bring on the notorious ailment. Genetically modified (GM) tobacco (Nicotiana tabacum cv. Petit Havana) harboring ompH(A:3) was constructed to develop a plant expression system for the protein, OmpH(A:3). Some 987 bp-long (ORF with the stop codon, TAA) of the ompH(A:3) excluding the nucleotide for signal peptide, was amplified by RT-PCR with the gene specific primers and pGEM-T-ompH(A:3) as template DNA. The PCR-amplified DNA was ligated into BamHI/Sacl-cut pBI121 to obtain a recombinant plasmid, pBI121-ompH(A:3). It was then transformed into Agrobacterium tumefaciens (LBA 4404) by liquid nitrogen method to generate a recombinant clone of Agrobacterium LBA4404/pBI121-ompH(A:3). The Agrobacterium LBA4404/pBI121-ompH(A:3) was inoculated into leaf discs of tobacco (2 day old). The gene-transfected leaves were cultured on Murashige-Skoog basal medium containing kanamycin (50 mg/mL) to generate numerous calli, from which some GM tobacco plants were obtained. Transgenicity of the tobacco plant was confirmed by PCR screening along with the DNA sequencing. Also, its expression in the GM-tobacco was examined qualitatively as well as quantitatively by ELISA/Western blot. These results suggest that the genetically modified tobacco plant can be potentially used as a model system to develop plant-based vaccine against the fowl cholera.     

Highly selective biotransformation of ginsenoside Rb<Subscript>1</Subscript> to Rd by the phytopathogenic fungus<Emphasis Type="Italic"> Cladosporium fulvum</Emphasis> (<Emphasis Type="Italic">syn</Emphasis>.<Emphasis Type="Italic"> Fulvia fulva</Emphasis>)

Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb₁ to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml⁻¹; temperature, 37°C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb₁ by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Identification of a novel two-partner secretion system from<Emphasis Type="Italic"> Burkholderia pseudomallei</Emphasis>

Two adjacent genes, bpaA and bpaB, whose products display significant similarity to a number of two-partner secretion (TPS) systems have been identified in Burkholderia pseudomallei strain 08, but are absent from the closely related avirulent species B. thailandensis. They possess a number of sequence features characteristic of TPS systems, including the presence of an NPNGI motif in a region of BpaA which strongly resembles a TPS secretion domain. BpaA is a very large protein (~530 kDa) and contains three repeats, each 600–800-amino acids long. Putative membrane-spanning regions in BpaB were identified through alignment with TpsB family members, and this also revealed an N-terminal extension not found in other TpsB proteins. The bpaA gene was found to be absent from the majority of B. pseudomallei strains. It appears that bpaAB are located within a putative genomic island that is inserted in close proximity to a methionine tRNA^CAT-encoding gene. Expression of BpaA was undetectable in cells grown in laboratory media. However, owing to the similarity of BpaA to known adhesin molecules, a potential role of BpaA in virulence was investigated in cell culture and in an animal model, but no evidence for such a role was found in these test systems.Communicated by W. Goebel