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Doctoral Thesis
DOI
10.11606/T.87.2010.tde-29042010-102817
Document
Author
Full name
Rogério de Sousa Gomes
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2010
Supervisor
Committee
Gomez, José Gregorio Cabrera (President)
Alterthum, Flavio
Baldini, Regina Lúcia
Barbosa, Heloiza Ramos
Vicente, Elisabete Jose
Title in Portuguese
Obtenção de mutantes deficientes no acúmulo de PHA e construção de linhagens recombinantes para o controle da composição monomérica
Keywords in Portuguese
3-Cetoacil-CoA redutase
PHA sintase
Polihidroxialcanoatos
Polímeros biodegradáveis
Pseudomonas
Transposon mini-Tn5
Abstract in Portuguese
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 β-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 β-oxidação para a síntese de PHA.
Title in English
Mutants deficient on polyhydroxyalkanoate (PHA) accumulation and construction of recombinants to control monomer composition on PHA
Keywords in English
Biodegradable Polymers
Ketoacyl-CoA reductase
Mini-Tn5 transposon
PHA synthase
Polyhydroxi alkanoates
Pseudomonas
Abstract in English
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 β-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 β-oxidation to PHA biosynthesis.
 
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Publishing Date
2010-05-19
 
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