Synthesis of polyhydroxyalkanoate in the peroxisome of Pichia pastoris

Polyhydroxyalkanoates (PHAs) are polyesters naturally produced by bacteria that have properties of biodegradable plastics and elastomers. A PHA synthase from Pseudomonas aeruginosa modified at the carboxy-end for peroxisomal targeting was transformed in Pichia pastoris. The PHA synthase was expressed under the control of the promoter of the P. pastoris acyl-CoA oxidase gene. Synthesis of up to 1% medium-chain-length PHA per g dry weight was dependent on both the expression of the PHA synthase and the presence of oleic acid in the medium. PHA accumulated as inclusions within the peroxisomes. P. pastoris could be used as a model system to study how peroxisomal metabolism needs to be modified to increase PHA production in other eukaryotes, such as plant

Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene

BACKGROUND: Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2(Ps) cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2(Ps) is limited with very low PHA yield. RESULTS: To improve the production of PHA with a wide range of monomer compositions in E. coli, a series of optimization strategies were applied on the PHA synthase PhaC2(Ps). Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli. The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2(Ps), the optimized PhaC2(Ps) increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2(Ps)O with a hairpin structure in the 5’ untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2(Ps). CONCLUSIONS: The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2(Ps). These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production

Metabolism of Poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. Identification and Sequences of Genes and Function of the Encoded Proteins in the Synthesis and Degradation of PHA

Pseudomonas oleovorans accumulates poly(3-hydroxyalkanoates) (PHAs) after growth on medium chain length hydrocarbons. Large amounts of this polyester are synthesized when cells are grown under nitrogen-limiting conditions. When nitrogen is resupplied in the medium, the accumulated PHA is degraded. In this paper, we describe mutants which are defective in the synthesis or in the degradation of PHA. These mutants were used to select DNA fragments which encode PHA polymerases and a PHA depolymerase. A 25-kilobase (kb) DNA fragment was isolated from P. oleovorans that complements a Pseudomonas putida mutant unable to accumulate PHA. Subcloning resulted in the assignment of a 6.4-kb EcoRI fragment as the pha locus, containing genetic information of PHA synthesis. Mutants in the PHA degradation pathway were also complemented by this fragment, indicating that genes encoding PHA biosynthetic and degradative enzymes are clustered. Analysis of the DNA sequence of the 6.4-kb fragment revealed the presence of two open reading frames encoding PHA polymerases based on homology to the poly(3-hydroxybutyrate) polymerase from Alcaligenes eutrophus. A third open reading frame complemented the PHA degradation mutation and is likely to encode a PHA depolymerase. The presence of two PHA polymerases is due to a 2098-base pair DNA duplication. The PHA polymerases are 53% identical and show 35-40% identity to the poly(3-hydroxybutyrate) polymerase. No clear difference in specificity was found for the PHA polymerases. However, with the pha locus cloned on a multicopy vector, a polymer was accumulated that contains a significantly higher amount of substrate-derived monomers. An increase in the rate of polyester synthesis versus oxidation of the monomers in the beta-oxidation explains these findings

Controlled biotechnological production of polyhydroxyalkanoates

Předložená diplomová práce se zabývá produkcí polyhydroxyalkanoátů (PHA) bakterií Cupriavidus necator H16. Cílem práce byla příprava, selekce a charakterizace mutantních kmenů schopných vyšší produkce PHA. V teoretické části byla zpracována literární rešerše zabývající se nejdůležitějšími typy PHA, bakterií Cupriavidus necator a způsoby indukce mutageneze. V experimentální části byly připraveny mutantní kmeny pomocí fyzikální a chemické mutageneze. Mutantní kmeny schopné nadprodukce PHA byly selektovány pomocí kultivace na minerálním médium s olejem. Pro další studium byly vybrány 4 mutantní kmeny schopné nadprodukce PHA. Tyto mutantní kmeny byly dále podrobeny biochemické charakterizaci. Byly naměřeny specifické aktivity vybraných intracelulárních enzymů včetně enzymů podílejících se na biosyntéze PHA. Také byla naměřena resistence mutantů vůči oxidačnímu stresu. Bylo zjištěno, že mutantní kmeny schopné nadprodukce PHA mají vyšší aktivity enzymů produkujících NADPH. NADPH je jeden z klíčových substrátů ovlivňujících směr toku acetyl-CoA metabolizmem. Vyšší intracelulární koncentrace NADPH parciálně inhibuje Krebsův cyklus a aktivuje akumulaci PHA. Aktivity acetoacetyl-CoA reduktázy a PHA syntázy, enzymů zapojených do syntézy PHA, těchto mutantů proto byly také vyšší stejně jako molekulová hmotnost připravených polymerů. Aplikace fyzikálních a chemických mutagenů je způsob, kterým lze připravit biotechnologicky perspektivní mutantní kmeny schopné nadprodukce PHA.This diploma thesis deals with production of polyhydroxyalkanoates (PHA) by bacterial strain Cupriavidus necator H16. Goal of this work was preparation, selection and characterization of mutant strains overproducing PHA. Theoretical focuses on the most important PHA, bacteria Cupriavidus necator and mutagenesis techniques. In practical part mutant strains were prepared through physical and chemical mutagenesis. Mutant strains overproducing PHA were selected by cultivation in mineral medium with oil. For further study, 4 mutant strains overproducing PHA were selected. These mutants were biochemically characterized. Specific activities of several intracellular enzymes including enzymes involved in PHA biosynthesis were measured. Resistance of mutants against oxidative stress was measured as well. Mutant strains overproducing PHA revealed higher enzymatic activities of NADPH producing enzymes. Generally, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH partially inhibits TCA cycle and activates accumulation of PHA. Therefore, activities of acetoacetyl-CoA reductase and PHB synthase, enzymes directly involved in PHA synthesis were higher as compared to wild strain as well as molecular weight of produced materials. It can be concluded that biotechnologically perspective mutagens capable of PHA overproduction can be prepared by application of chemical and physical mutagens.

Valorisation of local agro-industrial processing waters as growth media for polyhydroxyalkanoates (PHA) production

International audiencePolyhydroxyalkanoates (PHA) are bacterial polyesters usually produced from costly sugars or volatile fatty acids (VFAs). In this work, two processing waters rich in vegetable proteins and reducing sugars, i.e., a mixture of saccharose and stachyose in Leguminous Processing Water (LPW) and a mixture of glucose and fructose in Fruit Processing Water (FPW), were tested as growth medium for PHA production in a two-stage fermentation with a unique marine bacterial species: Halomonas i4786. In preliminary shake flask experiments, it was shown that the two media can effectively support the bacterial growth and the accumulation of PHA (evaluated using Nile Red staining). In batch cultivation mode in a 5-L fermentor, PHA productivities of 1.6 g L−1 and 1.8 g L−1 were further achieved within 72 h, in LPW and FPW respectively. Polymer characterization by Differential Scanning Calorimetry and Steric Exclusion Chromatography indicated that the two substrates led to the biosynthesis of polymers with different chain length, distribution and crystallinity. To summarize, these results show that by-products derived from local agri-food industry can be used as a user-adapted and cost-effective source to produce bio-sourced and biodegradable plastic material

Novel particulate vaccine candidates recombinantly produced by pathogenic and nonpathogenic bacterial hosts : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand.

Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized as small spherical cytoplasmic inclusion bodies by a range of bacteria. Recently, PHA beads have been investigated for use as a vaccine delivery platform by using engineered heterologous production hosts that allowed the efficient display of vaccine candidate antigens on the beads surface and were found to greatly improve immunogenicity of the displayed antigens. However, like other subunit vaccines, these antigen-displaying (vaccine) PHA beads only provide a limited repertoire of antigens. In this thesis we investigate the idea of directly utilizing the disease causative pathogen or model organism to produce vaccine PHA beads with a large antigenic repertoire. These beads are hypothesized to have the potential to induce greater protective immunity compared to production of the same PHA bead in a heterologous production host. This concept was exemplified with Pseudomonas aeruginosa and Mycobacterium tuberculosis as model human pathogens. For P. aeruginosa we describe the engineering of this bacterium to promote PHA and Psl (polysaccharide) production. This represents a new mode of functional display for the engineering, production, and validation of a novel OprI/F-AlgE fusion antigen-displayed on PHA beads. For the disease tuberculosis we investigated the use of nonpathogenic M. smegmatis as a model organism for M. tuberculosis. We described the bioengineering, production, and validation of Ag85AESAT- 6 displayed on PHA beads produced in M. smegmatis. Here we showed that both organisms were harnessed to produce custom-made PHA beads for use as particulate subunit vaccines that carried copurifying pathogen-derived proteins as a large antigenic repertoire and the ability of these vaccine PHA beads to generate a protective immune response. This novel bioengineering concept of particulate subunit vaccine production could be applied to a range of pathogens naturally producing PHA inclusions for developing efficacious subunit vaccines for infectious diseases

Production of Medium Chain Length Polyhydroxyalkanoates From Oleic Acid Using Pseudomonas Putida Pga1 by Fed Batch Culture

Bacterial polyhydroxyalkanoates (PHAs) are a class of polymers currently receiving much attention because of theirpotential as renewable and biodegradable plastics. A wide variety of bacteria has been reported to produce PHAsincluding Pseudomonas strains. These strains are known as versatile medium chain length PHAs (PHAs-mcl) producersusing fatty acids as carbon source. Oleic acid was used to produce PHAs-mcl using Pseudomonas putida PGA 1 bycontinuous feeding of both nitrogen and carbon source, in a fed batch culture. During cell growth, PHAs alsoaccumulated, indicating that PHA production in this organism is growth associated. Residual cell increased until thenitrogen source was depleted. At the end of fermentation, final cell concentration, PHA content, and productivity were30.2 g/L, 44.8 % of cell dry weight, and 0.188 g/l/h, respectively

Picosecond Laser Ablation of Polyhydroxyalkanoates (PHAs): Comparative Study of Neat and Blended Material Response

Polyhydroxyalkanoates (PHAs) have emerged as a promising biodegradable and biocompatible material for scaffold manufacturing in the tissue engineering field and food packaging. Surface modification is usually required to improve cell biocompatibility and/or reduce bacteria proliferation. Picosecond laser ablation was applied for surface micro structuring of short- and medium-chain length-PHAs and its blend. The response of each material as a function of laser energy and wavelength was analyzed. Picosecond pulsed laser modified the surface topography without affecting the material properties. UV wavelength irradiation showed halved ablation thresholds compared to visible (VIS) wavelength, revealing a greater photochemical nature of the ablation process at ultraviolet (UV) wavelength. Nevertheless, the ablation rate and, therefore, ablation efficiency did not show a clear dependence on beam wavelength. The different mechanical behavior of the considered PHAs did not lead to different ablation thresholds on each polymer at a constant wavelength, suggesting the interplay of the material mechanical parameters to equalize ablation thresholds. Blended-PHA showed a significant reduction in the ablation threshold under VIS irradiation respect to the neat PHAs. Picosecond ablation was proved to be a convenient technique for micro structuring of PHAs to generate surface microfeatures appropriate to influence cell behavior and improve the biocompatibility of scaffolds in tissue engineerin

Novel essential role of ethanol oxidation genes at low temperature revealed by transcriptome analysis in the antarctic bacterium pseudomonas extremaustralis

Temperature is one of the most important factors for bacterial growth and development. Cold environments are widely distributed on earth, and psychrotolerant and psychrophilic microorganisms have developed different adaptation strategies to cope with the stress derived from low temperatures. Pseudomonas extremaustralis is an Antarctic bacterium able to grow under low temperatures and to produce high amounts of polyhydroxyalkanoates (PHAs). In this work, we analyzed the genome-wide transcriptome by RNA deepsequencing technology of early exponential cultures of P. extremaustralis growing in LB (Luria Broth) supplemented with sodium octanoate to favor PHA accumulation at 8°C and 30°C. We found that genes involved in primary metabolism, including tricarboxylic acid cycle (TCA) related genes, as well as cytochromes and amino acid metabolism coding genes, were repressed at low temperature. Among up-regulated genes, those coding for transcriptional regulatory and signal transduction proteins were over-represented at cold conditions. Remarkably, we found that genes involved in ethanol oxidation, exaA, exaB and exaC, encoding a pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the cytochrome c550 and an aldehyde dehydrogenase respectively, were up-regulated. Along with RNA-seq experiments, analysis of mutant strains for pqqB (PQQ biosynthesis protein B) and exaA were carried out. We found that the exaA and pqqB genes are essential for growth under low temperature in LB supplemented with sodium octanoate. Additionally, prosaniline assay measurements showed the presence of alcohol dehydrogenase activity at both 8°C and 30°C, while the activity was abolished in a pqqB mutant strain. These results together with the detection of ethanol by gas chromatography in P. extremaustralis cultures grown at 8°C support the conclusion that this pathway is important under cold conditions. The obtained results have led to the identification of novel components involved in cold adaptation mechanisms in this bacterium, suggesting for the first time a role of the ethanol oxidation pathway for bacterial growth at low temperatures.Fil: Tribelli, Paula Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Solar Venero, Esmeralda Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Ricardi, Martiniano María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Gómez Lozano, Maria. Technical University of Denmark; DinamarcaFil: Raiger Iustman, Laura Judith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Molin, Søren. Technical University of Denmark; DinamarcaFil: López, Nancy Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin