This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.

Pseudomonas putida produz PHA de cadeia média (PHAMCL) a partir de carboidratos e óleos vegetais. Genes de biossíntese de P3HB (poli-3-hidroxibutirato) de Ralstonia eutropha foram inseridos em P. putida IPT046 selvagem e mutantes PHA-. A expressão de phaC, codificador da PHA sintase, permitiu o acumulo de PHA com alto teor de 3HB, indicando que P. putida possui enzimas geradoras de 3HB. A expressão de phaB mostrou que seu produto canaliza 3HB e 3-hidroxihexanoato (3HHx) da <font face=\"Symbol\">&#946-oxidação para a PHA sintase. Ao expressar phaC em IPT461, afetado na PHA sintase, 30% de P3HB-co-3HHx-co-3HO foram produzidos a partir de carboidratos, mostrando que há vias metabólicas eficientes para suprir 3HAMCL, que são incorporados pela PHA sintase de R. eutropha. Usando transposon mini-Tn5, obtiveram-se mutantes deficientes na biossíntese de PHA a partir de glicose ou glicose e octanoato. Não se detectaram mutantes exclusivamente deficientes no acúmulo de PHA a partir de octanoato, indicando que diferentes produtos gênicos canalizam intermediários da <font face=\"Symbol\">&#946-oxidação para a síntese de PHA. / Pseudomonas putida produces medium-chain-length PHA (PHAMCL) from carbohydrates and plant oils. Recombinants harboring the P3HB (polyhydroxybutyrate) biosynthesis genes from Ralstonia eutropha were constructed on P. putida IPT046 wild type and PHA- mutants. Expression of phaC, encoding, PHA synthase, allowed the synthesis of PHA with high 3HB content, indicating that P. putida has enzymes to generate 3HB. Expression of phaB showed that its product channels 3HB and 3-hydroxyhexanoate (3HHx) from <font face=\"Symbol\">&#946-oxidation to the PHA synthase. When phaC was expressed on IPT461, affected in the PHA synthase, 30% P3HB-co-3HHx-co-3HO were accumulated from carbohydrates, showing that there are metabolic pathways efficient to supply 3HAMCL, which are incorporated by the R. eutropha PHA synthase. Using mini-Tn5 transposon, mutants deficient on PHA biosynthesis from glucose or glucose and octanoate were produced. No mutant deficient exclusively on PHA biosynthesis from octanoate was detected. Thus different gene products may channel intermediates from <font face=\"Symbol\">&#946-oxidation to PHA biosynthesis.

Polyhydroxyalkanoates (PHAs) are polyesters composed of (R)-3-hydroxy-fatty acids. A variety of gram-positive as well as gram-negative bacteria and some archaea are able to produce these biopolymers as energy and carbon storage materials. In times of unbalanced growth, when carbon is available in excess but other nutrients are limited, PHA inclusions are formed. These granules are water-insoluble, stored intracellularly and can be maintained outside the cell as beads. The key enzyme for the formation of PHA inclusions is the PHA synthase PhaC, which catalyses the polymerization of (R)- 3-hydroxyacyl-CoA to PHA with the concomitant release of CoA. The PHA synthase from Ralstonia eutropha (currently Cupriavidus necator), which is covalently bound to the PHA granule surface, tolerates fusions to its N terminus without loss of activity. In this study it was investigated if it would also tolerate translational fusions to its C terminus. A specially designed linker was employed, aiming at maintaining the hydrophobic surroundings of the R. eutropha synthase C terminus to allow proper folding and activity. Two reporter proteins were tested as fusion partners, the maltose binding protein MalE and the green fluorescent protein GFP. As GFP is a hydrophobic protein itself, no additional linker between the PHA synthase and the reporter protein was necessary to produce PHA granules displaying the functional fusion protein on the surface. Principally, the PHA synthase PhaC tolerates translational fusions to its C terminus but the nature of the fusion partner influences the functionality. Recently, PHA granules have often been acknowledged as bio-beads. A one-step production allows the formation of functionalised beads without the need for further cross-linking to impart desired surface properties. PHA beads displaying a gold- or silica-binding peptide at the N terminus of PhaC were constructed and tested for their applicability. Additionally, these beads were able to bind IgG due to the ZZ domain of the IgG binding protein A, which was employed as a linker sequence. These functionalised beads can be used as molecular tools in bioimaging and biomedicine, combining organic core with inorganic-binding shell structures. In a different biomimetic approach, the display of ten lysine residues at the granule surface was achieved using the phasin protein PhaP as the anchoring matrix. Extensive work was performed in an attempt to also employ the synthase protein, but was unsuccessful. These positively charged bio-beads can be used for dispersion or crosslinking experiments as well as silica binding.

Pseudomonas putida produz PHA de cadeia média (PHAMCL) a partir de carboidratos e óleos vegetais. Genes de biossíntese de P3HB (poli-3-hidroxibutirato) de Ralstonia eutropha foram inseridos em P. putida IPT046 selvagem e mutantes PHA-. A expressão de phaC, codificador da PHA sintase, permitiu o acumulo de PHA com alto teor de 3HB, indicando que P. putida possui enzimas geradoras de 3HB. A expressão de phaB mostrou que seu produto canaliza 3HB e 3-hidroxihexanoato (3HHx) da <font face=\"Symbol\">&#946-oxidação para a PHA sintase. Ao expressar phaC em IPT461, afetado na PHA sintase, 30% de P3HB-co-3HHx-co-3HO foram produzidos a partir de carboidratos, mostrando que há vias metabólicas eficientes para suprir 3HAMCL, que são incorporados pela PHA sintase de R. eutropha. Usando transposon mini-Tn5, obtiveram-se mutantes deficientes na biossíntese de PHA a partir de glicose ou glicose e octanoato. Não se detectaram mutantes exclusivamente deficientes no acúmulo de PHA a partir de octanoato, indicando que diferentes produtos gênicos canalizam intermediários da <font face=\"Symbol\">&#946-oxidação para a síntese de PHA. / Pseudomonas putida produces medium-chain-length PHA (PHAMCL) from carbohydrates and plant oils. Recombinants harboring the P3HB (polyhydroxybutyrate) biosynthesis genes from Ralstonia eutropha were constructed on P. putida IPT046 wild type and PHA- mutants. Expression of phaC, encoding, PHA synthase, allowed the synthesis of PHA with high 3HB content, indicating that P. putida has enzymes to generate 3HB. Expression of phaB showed that its product channels 3HB and 3-hydroxyhexanoate (3HHx) from <font face=\"Symbol\">&#946-oxidation to the PHA synthase. When phaC was expressed on IPT461, affected in the PHA synthase, 30% P3HB-co-3HHx-co-3HO were accumulated from carbohydrates, showing that there are metabolic pathways efficient to supply 3HAMCL, which are incorporated by the R. eutropha PHA synthase. Using mini-Tn5 transposon, mutants deficient on PHA biosynthesis from glucose or glucose and octanoate were produced. No mutant deficient exclusively on PHA biosynthesis from octanoate was detected. Thus different gene products may channel intermediates from <font face=\"Symbol\">&#946-oxidation to PHA biosynthesis.

Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado. / The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.

Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado. / The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.