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RAVELLI, T. G. Selection of PHA synthases coding genes for the construction of recombinant Burkholderia sacchari and Pseudomonas sp and evaluation of polyhydroxyalkanoate production with different monomer compositions. 2014. 107 p. Masters thesis (Microbiology) – Instituto de Ciências Biomédicas, Universidade São Paulo, São Paulo, 2014. Polyhydroxyalkanoates (PHA) are polyesters accumulated by several bacteria from renewable sources that arouse great industrial interest because they are biodegradable and biocompatible thermoplastic. The variability of the PHA monomer composition determines its mechanical properties and allows their use in many applications. The PHAs may be formed by shortchain monomers (PHASCL) or medium (PHAMCL), or even form polymers (PHASCL coPHAMCL) having intermediate properties. Three aspects are crucial for the production of PHA: the carbon source, the bacterial metabolic pathways and the PHA synthase enzyme. The PHA synthase catalyzes the polymerization of R-hydroxyacyl-CoA molecules forming biopolymer, and thus, the key enzyme in the biosynthesis of PHA. Four types of PHA synthases are known according to their substrate specificity and the peptide composition. Many studies have been undertaken to different compositions of the polymer. Thus, the aim of this paper was to search for genes encoding this enzyme, recombinant construction and assessment carrying such genes to find materials with new properties and applications. Initially we searched for new PHA synthase genes from 390 clones of a metagenomic library by PCR as previously detected positive for any type of PHA synthase. Later, two new sources of PHA synthase were sought: genes encoding PHA synthase III Thiocapsa pfennigii and Allochromatium vinosum. Out of 390 clones, only 2 showed positive for PHA synthase class II by PCR and sequencing reaction, showing similarity to known genes. Because of the fact that the type II PHA synthases has already been widely studied, unlike those of the type III, most promising for this work, coding genes were sought to test their integration in host able to accumulate high concentrations of PHA, but affected in its native PHA synthase (Pseudomonas putida and Burkholderia sacchari), evaluating the type of PHA produced Thus, recombinant Pseudomonas sp and B. sacchari were built by the introduction of T. pfennigii genes (phaECTp) and C. vinosum (phaECCv ) cloned into pBBR1-MCS2. The PHA production by recombinant obtained was qualitatively assessed by staining tests with Sudan Black B, indicated positive results only in Burkholderia sacchari harboring the gene C. vinosum being that Pseudomonas putida for housing phaECCv and phaECTp and Burkholderia sacchari phaECTp the result was negative . Quantitatively, PHA production was assessed in shake flask tests, using glucose and fatty acids containing from 3 to 9 carbons. The expression of genes in B. sacchari phaECCv PHA was successful, since they were able to restore the production of PHA in the mutant. Using glucose, both the wild type strain and the recombinant Burkholderia sacchari produced a PHA composed of monomers of 3-hydroxybutyrate (3HB). From hexanoate and glucose gave a copolymer in which the 3HHx was increased fraction of 0.30 mol% in the wild type strain to 1.85 mol% in the mutant strain. Pseudomonas putida harboring phaECCv produced a PHA in which there was an increase in 3HHx fraction from 0 to 9.11 mol% when glucose was supplied as a carbon source. These lines can be used to produce PHA with varied composition, controlling the incorporation of 3HHx fractions which should reflect on their properties, expanding its application possibilities.

Seleção de genes codificadores de PHA sintases para a construção de recombinantes em Burkholderia sacchari e Pseudomonas sp e avaliação da produção de polihidroxialcanoatos com diferentes composições monoméricas.